Restoration of visual responses by in vivo delivery of rhodopsin nucleic acids

ABSTRACT

Nucleic acid vectors encoding light-gated cation-selective membrane channels, in particular channelrhodopsin-2 (Chop2), converted inner retinal neurons to photosensitive cells in photoreceptor-degenerated retina in an animal model. Such treatment restored visual perception and various aspects of vision. A method of restoring light sensitivity to a retina of a subject suffering from vision loss due to photoreceptor degeneration, as in retinitis pigmentosa or macular degeneration, is provided. The method comprises delivering to the subject by intravitreal or subretinal injection, the above nucleic acid vector which comprises an open reading frame encoding a rhodopsin, to which is operatively linked a promoter and transcriptional regulatory sequences, so that the nucleic acid is expressed in inner retinal neurons. These cells, normally light-insensitive, are converted to a light-sensitive state and transmit visual information to the brain, compensating for the loss, and leading to restoration of various visual capabilities.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was funded in part by grants from the National Institutesof Health grants (EY12180, EY-04068, EY16087, EY17130 and EY11522) whichprovides to the United States government certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention in the field of molecular biology and medicinerelates to the use of microbial-type rhodopsins, such as the light-gatedcation-selective membrane channel, channelrhodopsin-2 (Chop2) to convertinner retinal neurons to photosensitive cells inphotoreceptor-degenerated retina, thereby restoring visual perceptionand various aspects of vision.

2. Description of the Background Art

Vision normally begins when rods and cones, also called photoreceptors,convert light signals to electrical signals that are then relayedthrough second- and third-order retinal neurons and the optic nerve tothe lateral geniculate nucleus and, then to the visual cortex wherevisual images are formed (Baylor, D, 1996, Proc. Natl. Acad. Sci. USA93:560-565; Wassle, H, 2004, Nat. Rev. Neurosci. 5:747-57). For apatient who is vision-impaired due to the loss of photoreceptors, visualperception may be induced by providing electrical stimulation at one ofthese downstream neuronal locations, depending on the nature of theparticular impairment.

The severe loss of photoreceptor cells can be caused by congenitalretinal degenerative diseases, such as retinitis pigmentosa (RP) (Sung,C H et al., 1991, Proc. Natl. Acad. Sci. USA 88 :6481-85; Humphries, Pet al., 1992, Science 256:804-8; Weleber, R G et al., in: S J Ryan, Ed,Retina, Mosby, St. Louis (1994), pp. 335-466), and can result incomplete blindness. Age-related macular degeneration (AMD) is also aresult of the degeneration and death of photoreceptor cells, which cancause severe visual impairment within the centrally located best visualarea of the visual field.

Both rodents and humans go progressively blind because, as rods andcones are lost, there is little or no signal sent to the brain.Inherited retinal degenerations that cause partial or total blindnessaffect one in 3000 people worldwide. Patients afflicted with Usher'sSyndrome develop progressive deafness in addition to retinaldegeneration. There are currently no effective treatments or cures forthese conditions.

Basic research on approaches for retinal degeneration has long beenclassified into two approaches: (1) treatments to preserve remainingphotoreceptors in patients with retinal degenerative disease, and (2)methods to replace photoreceptors lost to retinal degeneration. Patientsafflicted with retinal disease often group themselves into those seekingways to slow the loss of their diminishing vision and those who arealready legally blind (“no light perception”), having lost theirphotoreceptors because of an inherited eye disease or trauma.

For the first approach, neuroprotection with neurotrophic factors(LaVail, M M et al., 1992, Proc. Natl. Acad. Sci. USA 89:11249-53) andvirus-vector-based delivery of wild-type genes for recessive nullmutations (Acland, G M et al., 2001, Nat. Genet. 28:92-95) have come thefurthest—to the point of a Phase I/II clinical trial (Hauswirth, W W,2005, Retina 25, S60; Jacobson, S, Protocol #0410-677, World Wide WebURL: webconferences.com/nihoba/16_jun_(—)2005.html) gaining approval inthe U.S. for adeno-associated viral (AAV)-mediated gene replacementtherapy for Leber's Congenital Amaurosis (LCA), a specific form ofretinal degeneration. Unfortunately, for patients in advanced stages ofretinal degeneration, this approach is not applicable, and thephotoreceptor cells must be replaced.

For replacement, one approach involves transplantation (replacement) ofnormal tissues or cells to the diseased retina. Another involveselectrical-stimulation of remaining non-visual neurons via retinalimplants in lieu of the lost photoreceptive cells (prostheticsubstitution). However, both methods face many fundamental obstacles.For example, for successful transplantation, the implanted tissue orcells must integrate functionally within the host retina. Theelectrical-stimulation approaches are burdened with mechanistic andtechnical difficulties as well as problems related to lack of long-termbiocompatibility of the implanted bionic devices. In summary, thereexist no effective vision-restoring therapies for inherited blindingdisease.

The present inventors' strategy as disclosed herein, requires a suitablemolecular “light-sensor.” Previous studies reported the heterologousexpression of Drosophila rhodopsin (Zemelman, B V et al., 2002, Neuron33:15-22) and, more recently, melanopsin, the putative photopigment ofthe intrinsic photosensitive retinal ganglion cells (Melyan, Z. et al.,2005, Nature 433:741-5; Panda, S. et al., 2005, Science 307:600-604;Qiu, X. et al., 2005, Nature 433:745-9). These photopigments, however,are coupled to membrane channels via a G protein signaling cascade anduse cis-isoforms of retinaldehyde as their chromophore. As a result,expression of multiple genes would be required to renderphotosensitivity. In addition, their light response kinetics is ratherslow. Recent studies aimed to improve the temporal resolution describedthe engineering of a light-sensitive K⁻ channel (Banghart et al., 2004,Nat. Neurosci. 7:1381-6), though this required introduction of anexogenous “molecular tether” and use of UV light to unblock the channel.This engineered channel was proposed to be potentially useful forrestoring light sensitivity in degenerate retinas, but its expressionand function in retinal neurons remain unknown.

The present invention makes use of microbial-type rhodopsins similar tobacteriorhodopsin (Oesterhelt, D et al., 1973, Proc. Natl. Acad. Sci.USA 70:2853-7), whose conformation change is caused by reversiblephotoisomerization of their chromophore group, the all-trans isoform ofretinaldehyde, and is directly coupled to ion movement through themembrane (Oesterhelt, D., 1998, Curr. Opin. Struct. Biol. 8:489-500).Two microbial-type opsins, channelopsin-1 and -2 (Chop1 and Chop2), haverecently been cloned from Chlamydomonas reinhardtii (Nagel, G. et al.,2002, Science 296:2395-8; Sineshchekov, O A et al., 2002, Proc. Natl.Acad. Sci. USA 99:8689-94; Nagel, G. et al., 2003, Proc. Natl. Acad.Sci. USA 100, 13940-45) and shown to form directly light-gated membranechannels when expressed in Xenopus laevis oocytes or HEK293 cells in thepresence of all-trans retinal. Chop2, a seven transmembrane domainprotein, becomes photo-switchable when bound to the chromophoreall-trans retinal. Chop2 is particularly attractive because itsfunctional light-sensitive channel, channelrhodopsin-2 (Chop2retinalidene abbreviated ChR2) with the attached chromophore ispermeable to physiological cations. Unlike animal rhodopsins, which onlybind the 11-cis conformation, Chop2 binds all-trans retinal isomers,obviating the need for the all-trans to 11-cis isomerization reactionsupplied by the vertebrate visual cycle. However, the long-termcompatibility of expressing ChR2 in native neurons in vivo in generaland the properties of ChR2-mediated light responses in retinal neuronsin particular remained unknown until the present invention.

The present strategy is feasible because histological studies, both inanimal models of photoreceptor degeneration (Chang, B. et al., 2002,Vision Res. 42:517-25; Olshevskaya, E V et al., 2004, J. Neurosci.24:6078-85) and in postmortem patient eyes with almost completephotoreceptor loss due to RP (Santos, A H et al., 1997, Arch. Ophthalmol115:511-15; Milam, A H et al., 1998, Prog. Retin. Eye Res. 1 7:175-205),reported the preservation of a significant number of inner retinalneurons.

Retinal gene therapy has been considered a possible therapeutic optionfor man. For example, U.S. Pat. No. 5,827,702 refers to methods forgenerating a genetically engineered ocular cell by contacting the cellwith an exogenous nucleic acid under conditions in which the exogenousnucleic acid is taken up by the cell for expression. The exogenousnucleic acid is described as a retrovirus, an adenovirus, anadeno-associated virus or a plasmid. See, also, WO 00/15822 (Mar. 23,2000) and WO 98/48097 (Oct. 29, 1998)

Efforts in such gene therapy have focused mainly on slowing down retinaldegeneration in rodent models of primary photoreceptor diseases. Normalgenes and mutation-specific ribozymes delivered to photoreceptors haveprolonged the lifetime of these cells otherwise doomed for apoptoticcell death (Bennett, J., et al. 1996 Nat. Med. 2, 649-54; Bennett, J.,et al. 1998, Gene Therapy 5, 1156-64; Kumar-Singh, R et al., 1998 Hum.Mol. Genet. 7, 1893-900; Lewin, A S et al. 1998, Nat. Med. 4, 967-71;Ali, R et al. 2000, Nat. Genet. 25, 306-10; Takahashi, M. et al., 1999,J Virol. 73, 7812-6; Lau, D., et al., 2000, Invest. Ophthalmol. Vis.Sci. 41, 3622-33; and LaVail, M M, et al. 2000, Proc Natl Acad Sci USA97, 11488-93).

Retinal gene transfer of a reporter gene, green fluorescent protein(GFP), using a recombinant adeno-associated virus (rAAV) wasdemonstrated in normal primates (Bennett, J et al. 1999 Proc. Natl.Acad. Sci. USA 96, 9920-25). However, the restoration of vision in ablinding disease of animals, particularly in humans and other mammals,caused by genetic defects in retinal pigment epithelium (RPE) and/orphotoreceptor cells has not been achieved. Jean Bennett and colleagueshave described the rescue of photoreceptors using gene therapy in amodel of rapid degeneration of photoreceptors using mutations of theRP65 gene and replacement therapy with the normal gene to replace orsupplant the mutant gene. See, for example, US Patent Publication2004/0022766 of Acland, Bennett and colleagues. This therapy showed somesuccess in a naturally-occurring dog model of severe disease of retinaldegenerations—the RPE65 mutant dog, which is analogous to human LCA.

Advantages of the present approach include the fact that it does notrequire introducing exogenous cells and tissues or physical devices,thus avoiding many obstacles encountered by existing approaches; thepresent invention is applicable for the reversal of vision loss orblindness caused by many retinal degenerative diseases. By expressingphotosensitive membrane-channels or molecules in surviving retinalneurons of the diseased retina by viral based gene therapy method, thepresent invention can produce permanent treatment of the vision loss orblindness with high spatial and temporal resolution for the restoredvision.

To the extent that any specific disclosure in the aforementionedpublications or other publications may be considered to anticipate anygeneric aspect of the present invention, the disclosure of the presentinvention should be understood to include a proviso or provisos thatexclude of disclaim any such species that were previously disclosed. Theaspects of the present invention which are not anticipated by thedisclosure of such publications are also unobvious from the disclosureof these publications, due at least in part to the unexpectedly superiorresults disclosed or alleged herein.

SUMMARY OF THE INVENTION

The present invention is directed to the genetic conversion of survivinglight-insensitive inner retinal neurons in a retina in whichphotoreceptors are degenerating or have already died, into directlyphotosensitive neuronal cells, thereby imparting light sensitivity tosuch retinas and restoring one or more aspects of visual responses andfunctional vision to a subject suffering from such degeneration. Byrestoring light sensitivity to a retina lacking this capacity, due todisease, the invention provides a mechanism for the most basiclight-responses that are required for vision. Said another way, thepresent invention introduces a “light sensors” into retinal neurons thatnormally do not have them, to compensate for loss of retinalphotoreceptor cells.

The present inventors and colleagues investigated the feasibility ofusing Chop2/ChR2 to restore light sensitivity to the retinas that haveundergone rod and cone degeneration. The results presented herein showlong-term expression of Chop2/ChR2 in rodent inner retinal neurons invivo. The results also show that these inner retinal neurons can expressa sufficient number of functional ChR2 channels to produce robustmembrane depolarization or action potential firing without an exogenoussupply of all-trans retinal. Furthermore, the present inventorsdemonstrated that the expression of ChR2 in a photoreceptor-deficientmouse model not only enables retinal ganglion cells to encode lightsignals but also restores visually evoked responses in the visualcortex.

The present invention is directed to the restoration of vision loss toindividuals that have lost vision or are blind as a result of retinalphotoreceptor degeneration. The invention enables retinal neurons insuch a diseased retina to respond to light by expressing photosensitivemembrane-channels or molecules in these retinal neurons. Preferred thelight-sensitive channels or molecules are microbial type light-gatechannel rhodopsins, such as ChR2, ChR1, light-driven ion pump, such asbacteriorhodopsins (Lanyi, J K, 2004, Annu Rev Physiol. 66:665-88),halorhodopsins (Lanyi, J K, 1990, Physiol Rev. 70:319-30), and theirderivatives

As discovered by the present inventors, retinal neurons that arenormally not light sensitive (directly) in the retinas of blind mice,such as retinal ganglion cells (RGCs) and bipolar cells, can respond tolight when a green algae protein called channelrhodopsin-2 (ChR2), or abiologically active fragment or a conservative amino acid substitutionvariant thereof, is inserted into the neuronal cell membranes. The studywas conducted with mice that had been genetically bred to lose rods andcones, the light-sensitive cells in the retina, a condition that modelsRP in humans. In addition to RP, there are many forms of retinaldegenerative eye diseases that possibly could be treated by the presentapproach.

As disclosed herein, visual function can be restored by conveyinglight-sensitive properties to other surviving cells in the retina afterthe rods and cones have died. Using a DNA transfer approach, the presentinventors introduced the light-absorbing protein ChR2 into the mouseretinal neurons that survived after the rods and cones had died. Thesecells became light sensitive and sent signals via the optic nerve andhigher order visual pathways to the visual cortex where visualperception occurs. Using electrophysiologic means, it was shown that thesignals reached the visual cortex in a majority of the ChR2-treatedmice. The light sensitivity persisted for at least six months,suggesting that the subject might regain usable vision with additionalmaneuvers disclosed herein, such as expressing ChR2 in other types ofretinal cells or modifying the light sensitivity and/or wavelengthselectivity of ChR2, or using similar microbial proteins, to producediverse light-sensitive channels to improve outcomes for the restorationof normal vision.

As noted by persons of skill in this art, this strategy represents a“paradigm shift in the field” referring to a “new field ofre-engineering retinal interneurons as genetically modified ‘prosthetic’cells,” The present invention “opened the possibility of geneticallymodifying the surviving retinal interneurons to function as areplacement light-sensing receptor,” (Flannery, J and Greenberg, K.,2006, Neuron. 50:1-3; written as a preview to a publication in the sameissue of the present inventors and colleagues, Bi J. et al., Neuron 50,23-33, 2006).

The present inventors capitalized upon advancements in the field byusing viral vectors to transfer genes to retinal photoreceptor cells(Flannery J G et al., 1997, Proc. Natl. Acad. Sci. USA 94:6916-21). Theconversion of light-insensitive retinal interneurons into photosensitivecells introduces an entirely new direction for treatments of blindingretinal degeneration.

In one embodiment of the present invention, retinal bipolar cells,certain amacrine cells and ganglion cells are targeted for transductionof the Chop2 DNA, to convert them functionally into photosensitive cellsthat subsume the function of rods and cones. The layering of cells inthe retina is such that photoreceptor cells excite bipolar cells whichexcite ganglion cells to transmit signals to the visual cortex. It ispreferred to express the channel opsin of the present invention inbipolar ON-type cells. Intravitreal and/or subretinal injections areused to deliver DNA molecules and virus vectors to reach the cells beingtargeted.

In one embodiment, the promoter is from a mGluR6 promoter-region of theGrm6 gene (GenBank accession number BC041684), a gene that controlsexpression of metabotropic glutamate receptor 6 ((Ueda Y et al., 1997, JNeurosc 17:3014-23). The genomic sequence is shown in GenBank accessionnumber—AL627215. A preferred example of this promoter region sequencefrom the above GenBank record is SEQ ID NO:9 consisting of 11023nucleotides—as shown in FIG. 8. The original Umeda et al., studyemployed a 10 kb promoter, but the actual length of the promoter and thesequence that comprises control elements of Grm6 can be adjusted byincreasing or decreasing the fragment length. It is a matter of routinetesting to select and verify the action of the optimally sized fragmentfrom the Grm6 gene that drives transgenic expression of a selectedcoding sequence, preferably Chop2, in the desired target cells,preferably in bipolar cells which are rich in glutamate receptors,particularly the “on” type bipolar cells, which are the most bipolarcells in the retina (Nakajima, Y., et al., 1993, J Biol Chem268:11868-73).

The present invention is directed to a method of restoring lightsensitivity to a retina, comprising:

-   (a) delivering to retinal neurons a nucleic acid expression vector    that encodes a light-gated channel rhodopsin or a light-driven ion    pump rhodopsin expressible in the neurons, which vector comprises an    open reading frame encoding the rhodopsin, and operatively linked    thereto, a promoter sequence, and optionally, transcriptional    regulatory sequences; and-   (b) expressing the vector in the neurons, thereby restoring light    sensitivity.

The rhodopsin is preferably channelrhodopsin-2 (Chop2) or a biologicallyactive fragment or conservative amino acid substitution variant thereof.

The vector is preferably a rAAV viral vector.

The promoter may be a constitutive promoter such as a hybrid CMVenhancer/chicken β-actin promoter (CAG) (as indicated below as part ofSEQ ID NO: 1), or a CMV promoter. The promoter may also be (i) aninducible or (ii) a cell type-specific promoter, preferred examples ofthe latter being the mGluR6 promoter (e.g., part of a promoter sequenceSEQ ID NO:9), a Pcp2 (L7) promoter or a neurokinin-3 (NK-3) promoter.

A preferred vector in the above method comprises the CAG promoter, awoodchuck posttranscriptional regulatory element (WPRE), and a bovine orhuman growth hormone polyadenylation sequence.

In the present method, the retinal neurons are selected from ON- andOFF-type retinal ganglion cells, retinal rod bipolar cells, All amacrinecells and ON and OFF retinal cone bipolar cells. Preferably, the vectoris targeted to and expressed in ON type ganglion cells and/or ON typebipolar cells If the vector comprises the NK-3 promoter, the vector ispreferably targeted to OFF cone bipolar cells.

The invention is also directed to method of restoring photosensitivityto retinal neurons of a subject suffering from vision loss or blindnessin whom retinal photoreceptor cells are degenerating or have degeneratedand died, which method comprises:

-   (a) delivering to the retina of the subject a nucleic acid vector    that encodes a light-gated channel rhodopsin or a light-driven ion    pump rhodopsin expressible in the neurons, which vector comprises an    open reading frame encoding the rhodopsin, and operatively linked    thereto, a promoter sequence, and optionally, transcriptional    regulatory sequences;-   (b) expressing the vector in the neurons, wherein the expression of    the rhodopsin renders the neurons photosensitive, thereby restoring    of photosensitivity to the retina.

In this method the rhodopsin is preferably Chop2 or a biologicallyactive fragment or conservative amino acid substitution variant thereof.The vector is preferably a rAAV viral vector. Preferred promoters are asdescribed above for the above-presented embodiment. Preferred targetcells for the vector are as described above.

The restoration of photosensitivity using the above method preferablyresults in restoration of vision in the subject. The vision ispreferably measured by one or more of the following methods:

-   (i) a light detection response by the subject after exposure to a    light stimulus-   (ii) a light projection response by the subject after exposure to a    light stimulus;-   (iii) light resolution by the subject of a light versus a dark    patterned visual stimulus;-   (iv) electrical recording of a response in the visual cortex to a    light flash stimulus or a pattern visual stimulus

In this foregoing method, the vision loss or blindness may be a resultof a degenerative disease, preferably, retinitis pigmentosa orage-related macular degeneration.

In another embodiment, the subject is also provided with a visualprosthesis before, at the same time as, or after delivery of the vector.Preferred visual prostheses comprise retinal implants, corticalimplants, lateral geniculate nucleus implants, or optic nerve implants.

When employing the foregoing method, the subject's visual response maybe subjected to training using one or more visual stimuli. The trainingis preferably achieved by one or more of the following methods:

-   (a) habituation training characterized by training the subject to    recognize (i) varying levels of light and/or pattern stimulation,    and/or (ii) environmental stimulation from a common light source or    object; and-   (b) orientation and mobility training characterized by training the    subject to detect visually local objects and move among the objects    more effectively than without the training.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1I. Expression of Chop2-GFP in Retinal Neurons In vivo. FIG. 1Ashows the rAAV-CAG-Chop2-GFP-WPRE expression cassette. CAG: a hybrid CMVenhancer/chicken β-actin promoter. WPRE: woodchuck posttranscriptionalregulatory element. BGHpA: a bovine growth hormone polyadenylationsequence. (FIGS. 1B and 1C) Chop2-GFP fluorescence viewed in low (FIG.1B) and high (FIG. 1C) magnifications from eyes two months after theviral vector injection. (FIG. 1D) Confocal images of a ganglion cell,which show a stacked image (left) and a single optical section image(right). (FIG. 1E) Chop2-GFP fluorescence in a horizontal cell, whichshows GFP in soma, axon, and distal axon terminal. (FIGS. 1F and 1G)Chop2-GFP fluorescence in amacrine cells (FIG. 1F) and a retinal bipolarcell (FIGS. 1G). FIGS. 1H and 1I show fluorescence image (FIG. 1H) andphase contrast image (FIG. 1I) taken from a retina 12 months after theinjection of Chop2-GFP viral vectors. Images in (FIGS. 1B-1E) were takenfrom flat whole-mounts of rat retinas. Images in (FIGS. 1F-1I) weretaken from vertical slice sections of rat retinas. Scale bar: 200 μm in(FIG. 1B); 100 μm in (FIG. 1C); 15 μm in (FIG. 1D); 50 μm in (FIG. 1E),FIG. 1H), and (FIG. 1I); 25 μm in (FIG. 1F) and (FIG. 1G). ONL: outernuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer;GCL:

FIGS. 2A-2H. Properties of Light-Evoked Currents of the ChR2-expressingretinal neurons. (FIG. 2A) Phase contrast image (left) and fluorescenceimage (right) of a GFP-positive retinal neuron dissociated from theviral vector injected eye. Scale bar: 25 μm. (FIG. 2B) A recording ofChop2-GFP fluorescent retinal cell to light stimuli of wavelengthsranging from 420 to 580 nm. The light intensities were ranging from1.0-1.6×10¹⁸ photons cm ² S 1. (FIG. 2C) A representative recording ofthe currents elicited by light stimuli at the wavelength of 460 nm withlight intensities ranging from 2.2×10¹⁵ to 1.8×10¹⁸ photons cm⁻²s⁻¹.(FIG. 2D) Current traces after the onset of the light stimulation fromFIG. 2C shown in the expanded time scale. The line shows the fitting ofone current trace by an exponential function:I_((t))=a₀+a₁×(1−exp[−t/τ₁])+a₂×(exp[−t/τ₂]), in which τ₁ and τ₂represent the activation and inactivation time constant, respectively.(FIG. 2E) Current traces after the termination of the light stimulationfrom FIG. 2C shown in the expanded time scale. The line shows thefitting of one current trace by a single exponential function:I_((t))=a₀+a₁×(exp[−t/τ]), in which τ represent the deactivation timeconstant. (FIG. 2F) Light-intensity response curve. The data points werefitted with a single logistic function curve. (FIGS. 2F and H) Therelationships of light-intensity and activation time constant (FIG. 2G)and light-intensity and inactivation time constant (FIG. 2H) obtainedfrom the fitting shown in FIG. 2D. All recordings were made at theholding potential of −70 mV. The data points in FIG. 2F-2H are shown asmean±SD (n=7).

FIGS. 3A-3C. Properties of Light-Evoked Voltage Responses ofChR2-Expressing Retinal Neurons. (FIG. 3A) A representative recordingsfrom GFP-positive nonspiking neurons. The voltage responses wereelicited by four incremental light stimuli at the wavelength of 460 nmwith intensities ranging from 2.2×10¹⁵ to 1.8×10¹⁸ photons cm⁻²s⁻¹ incurrent clamp. The dotted line indicates the saturated potential level.(FIG. 3B) A representative recording from GFP-positive nonspikingneurons to repeat light stimulations. The light-evoked currents (toptraces) and voltage responses (bottom traces) from a same cells wereshown. Left panel shows the superimposition of the first (red) andsecond (black) traces in an expanded time scale. The dotted lineindicates the sustained component of the currents (top) and plateaumembrane potential (bottom). (FIG. 3C) A representative recording ofGFP-positive spiking neurons to repeated light stimulations. Theresponses in FIGS. 3B and 3C were evoked by light at the wavelength of460 nm with the intensity of 1.8×10¹⁸ photons cm⁻²s⁻¹.

FIGS. 4A-4I. Expression and Light-Response Properties of ChR2 in RetinalNeurons of rd1/rd1 Mice. (FIG. 4A) Chop2-GFP fluorescence viewed in flatretinal whole-mount of a 15 month old mouse with the Chop2-GFP viralvector injection at 9 months of age. (FIG. 4B) Chop2-GFP fluorescenceviewed in vertical section from the retina of a 6 month old mouse withthe injection of Chop2-GFP viral vectors at 3 months of age. (FIG. 4C)Light microscope image of a semithin vertical retinal section from a 5month old mouse (Chop2-GFP viral vectors injected at postnatal day 1).Scale bar: 50 μm in (FIG. 4A) and 30 μm in (FIGS. 4B and 4C). (FIGS.4D-4E) show representative recordings of transient spiking (FIG. 4D) andsustained spiking (FIG. 4E) GFP-positive neurons. The responses wereelicited by light of four incremental intensities at the wavelength of460 nm. The light intensity without neutral density (Log I=0) was3.6×10¹⁷ photons cm⁻² s⁻¹. The currents were recorded at the holdingpotential of −70 mV. The superimposed second (solid black) and fourth(dashed or red) current and voltage traces are shown in the right panelin the expanded time scale. (FIGS. 4F-4I) show the relationships of theamplitude of current (FIG. 4F), membrane depolarization (FIG. 4G), thenumber of spikes (FIG. 4H), and the time to the first spike peak (FIG.4I) to light intensity. Recordings were made from rd1/rd1 mice at ≧4months of age. The data points are the mean±SE (n=6 in FIG. 4F-4H andn=4 in FIG. 4I).

FIG. 5A-5D. Multielectrode Array Recordings of the ChR2-ExpressingRetinas of rd1/rd1 Mice. (FIG. 5A) A sample recording of light-evokedspike activities from the retinas of rd1/rd1 mice (ages≧4 months). Therecording was made in the present of CNQX (25 μM) and AP5 (25 μM).Prominent light-evoked spike activity was observed in 49 out of 58electrodes (electrode 15 was for grounding and electrode 34 wasdefective). (FIG. 5B) Sample light-evoked spikes recorded from a singleelectrode to three incremental light intensities. (FIG. 5C) The rasterplots of 30 consecutive light-elicited spikes originated from a singleneuron. (FIG. 5D) The averaged spike rate histograms. The lightintensity without neutral density filters (Log I=0) was 8.5×10¹⁷ photonscm⁻²s⁻¹. The responses shown in FIG. 5A were elicited by a single lightpulse without neutral density filters.

FIG. 6A-6E. Central Projections of Chop2-GFP-Expressing Retinal GanglionCells and Visual-Evoked Potentials in rd1/rd1 Mice. (FIG. 6A) GFPlabeled terminal arbors of retinal ganglion cells in ventral lateralgeniculate nucleus and dorsal lateral geniculate nucleus. (FIG. 6B)GFP-labeled terminal arbors of retinal ganglion cells in superiorcolliculus. OT: optical track; vLGN: ventral lateral geniculate nucleus;dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus. Scalebar: 200 μm in FIG. 6A), 100 μm in FIG. 6B). (FIG. 6C) VEPs recordedfrom a wild-type mouse. The responses were observed both to thewavelengths of 460 and 580 nm. (FIG. 6D) VEPs recorded from an rd1/rd1mouse injected with Chop2-GFP viral vectors. The responses were elicitedonly by light at the wavelength of 460 nm but not at the wavelength of580 nm. (FIG. 6E) No detectable VEPs were observed from rd1/rd1 miceinjected with viral vectors carrying GFP alone. The light intensitiesmeasured at the corneal surface at the wavelengths of 460 and 580 nmwere 5.5×10¹⁶ and 5.2×10¹⁶ photons cm⁻²s⁻¹, respectively. (FIG. 6F) Plotof the amplitude of VEPs from rd1/rd1 mice injected with Chop2-GFP viralvectors to various light intensities at the wavelengths of 420, 460,500, 520, and 540 nm. For each eye, the responses are normalized to thepeak response obtained at 460 nm. The data are the mean±SD (n=3 eyes).Spectral sensitivity at each wavelength was defined as the inverse ofthe interpolated light intensity to produce 40% of the normalized peakresponse, as indicated by the dot line. (FIG. 6G) The sensitivity datapoints were fitted by a vitamin-A₁-based visual pigment template with apeak wavelength of 461 nm.

FIG. 7 shows a map of the viral expression constructrAAV2-CAG-Chop2-GFP-WPRE (SEQ ID NO: 1), which comprises a Chop2-GFPfragment, an operatively linked a hybrid CMV enhancer/chicken β-actinpromoter (CAG), a woodchuck posttranscriptional regulatory element(WPRE), and a bovine growth hormone (BGH) polyadenylation sequence.

FIG. 8 (sheets 1-3) presents the sequence (SEQ ID NO:9)—11023 nt's—ofthe mGluR6 promoter region of the Grm6 gene (GenBank No. BC041684). Thegenomic sequence is provided in GenBank No. AL627215.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for treating an ocular disorder in ahuman, other mammalian or other animal subject. In particular, theocular disorder is one which involves a mutated or absent gene in aretinal pigment epithelial cell or a photoreceptor cell. The method ofthis invention comprises the step of administering to the subject byintravitreal or subretinal injection of an effective amount of arecombinant virus carrying a nucleic acid sequence encoding an ocularcell-specific normal gene operably linked to, or under the control of, apromoter sequence which directs the expression of the product of thegene in the ocular cells and replaces the lack of expression orincorrect expression of the mutated or absent gene.

Ocular Disorders

The ocular disorders for which the present methods are intended and maybe used to improve one or more parameters of vision include, but are notlimited to, developmental abnormalities that affect both anterior andposterior segments of the eye. Anterior segment disorders includeglaucoma, cataracts, corneal dystrophy, keratoconus. Posterior segmentdisorders include blinding disorders caused by photoreceptor malfunctionand/or death caused by retinal dystrophies and degenerations. Retinaldisorders include congenital stationary night blindness, age-relatedmacular degeneration, congenital cone dystrophies, and a large group ofretinitis-pigmentosa (RP)-related disorders. These disorders includegenetically pre-disposed death of photoreceptor cells, rods and cones inthe retina, occurring at various ages. Among those are severeretinopathies, such as subtypes of RP itself that progresses with ageand causes blindness in childhood and early adulthood and RP-associateddiseases, such as genetic subtypes of LCA, which frequently results inloss of vision during childhood, as early as the first year of life. Thelatter disorders are generally characterized by severe reduction, andoften complete loss of photoreceptor cells, rods and cones. (Trabulsi, EI, ed., Genetic Diseases of the Eye, Oxford University Press, NY, 1998).

In particular, this method is useful for the treatment and/orrestoration of at least partial vision to subjects that have lost visiondue to ocular disorders, such as RPE-associated retinopathies, which arecharacterized by a long-term preservation of ocular tissue structuredespite loss of function and by the association between function lossand the defect or absence of a normal gene in the ocular cells of thesubject. A variety of such ocular disorders are known, such as childhoodonset blinding diseases, retinitis pigmentosa, macular degeneration, anddiabetic retinopathy, as well as ocular blinding diseases known in theart. It is anticipated that these other disorders, as well as blindingdisorders of presently unknown causation which later are characterizedby the same description as above, may also be successfully treated bythis method. Thus, the particular ocular disorder treated by this methodmay include the above-mentioned disorders and a number of diseases whichhave yet to be so characterized.

Visual information is processed through the retina through two pathways:an ON pathway which signals the light ON, and an OFF pathway whichsignals the light OFF (Wassle, supra). It is generally believed that theexistence of the ON and OFF pathway is important for the enhancement ofcontrast sensitivity. The visual signal in the ON pathway is relay fromON-cone bipolar cells to ON ganglion cells. Both ON-cone bipolar cellsand ON-ganglion cells are depolarized in response to light. On the otherhand, the visual signal in the OFF pathway is carried from OFF-conebipolar cells to OFF ganglion cells. Both OFF-cone bipolar cells andOFF-ganglion cells are hypopolarized in response to light. Rod bipolarcells, which are responsible for the ability to see in dim light(scotopic vision), are ON bipolar cells (depolarized in response tolight). Rod bipolar cells relay the vision signal through AII amacrinecells (an ON type retinal cells) to ON an OFF cone bipolar cells.

The present Examples show functional consequence of expressingubiquitously expressing light sensitive channels, namely ChR2, inretinal ganglion cells by CAG promoter, and suggest that this sufficientfor restoring useful vision. However, targeting of depolarizing membranechannels, such as ChR2, to the ON-type retinal neurons might result inbetter useful vision. In addition, expression of light sensors in moredistal retinal neurons, such as bipolar cells, would utilize theremaining signal processing functions of the degenerate retina.Furthermore, by expressing a depolarizing light sensor, such as ChR2, inON type retinal neurons (ON type ganglion cells and/or ON type bipolarcells) and expressing a hypopolarizing light sensor, such ashalorhodopsin (a chloride pump) (Han, X et al., 2007, PLoS ONE, March21;2:e299; Zhang, F et al., 2007; Nature 446:633-9; present inventors'results) in OFF type retinal neurons (OFF type ganglion cells and/or OFFtype bipolar cells) could create ON and OFF pathways in photoreceptordegenerated retinas.

An alternative approach to restore ON and OFF pathways in the retina isachieved by, expressing a depolarizing light sensor, such as ChR2, torod bipolar cells or AII amacrine. This is because the depolarization ofrod bipolar cells or AII amacrine cells can lead to the ON and OFFresponses at the levels of cone bipolar cells and the downstream retinalganglion cells and, thus, the ON and OFF pathways that are inherent inthe retina could be maintained (Wässle, 2004).

According to the present invention, the followings approaches are usedto restore the light sensitivity of inner retinal neurons:

(1) Ubiquitously expressing light sensitive channels, such as ChR2, areemployed to produced membrane depolarization in all types of ganglioncells (both ON and OFF ganglion cells), or all types of bipolar cells(rod bipolar cells, and ON and OFF cone bipolar cells). The AAV vectorwith CAG promoter has already partially achieved this approach in rodentretinas, as exemplified herein.

(2) A depolarizing light sensor, such as ChR2, is targeted to ON typeretinal neurons such as ON type ganglion cells or ON type bipolar cells.A study from Dr. J. G. Flannery's group has identified the fragments ofa human gap junctional protein (connexin-36) promoter to target GFP inON-type retinal ganglion cells by using AAV-2 virus vector (Greenberg KP et al., 2007, In vivo Transgene Expression in ON-Type Retinal GanglionCells: Applications to Retinal Disease. ARVO abstract, 2007). A readilypackable shorter version of mGluR6 promoter of (<2.5 kb) would allowtargeting of ChR2 to ON type bipolar cells (both rod bipolar cells andON type cone bipolar cells).

(3) Cell specific promoters are used to target the specific types ofretinal neurons. A promoter that could target rod bipolar cells is Pep2(L7) promoter (Tomomura, M et al., 2001, Eur J Neurosci. 14:57-63). Thelength of the active promoter is preferably less that 2.5 Kb so it canbe packaged into the AAV viral cassette.

(4) A depolarizing light sensor, such as ChR2, is targeted to ON typeganglion cells or ON type cone bipolar cells and a hypopolarizing lightsensor, such as halorhodopsin, to OFF type ganglion cells or OFF typecone bipolar cells to create ON and OFF pathways. As described above, anadequately short (packable) version of mGluR6 promoter (<2.5 kb) wouldallow targeting of ChR2 to ON type bipolar cells. The Neurokinin-3(NK-3) promoter would be used to target halorhodopsin to OFF conebipolar cells (Haverkamp, S et al., 2002, J Comparative Neurology,455:463 - 76.

Vectors

According to the various embodiments of the present invention, a varietyof known nucleic acid vectors may be used in these methods, e.g.,recombinant viruses, such as recombinant adeno-associated virus (rAAV),recombinant adenoviruses, recombinant retroviruses, recombinantpoxviruses, and other known viruses in the art, as well as plasmids,cosmids and phages, etc. Many publications well-known in the art discussthe use of a variety of such vectors for delivery of genes. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, latest edition; Kay, M A. et al., 2001, Nat. Med.,7:33-40; and Walther W et al., 2000, Drugs 60:249-71).

Methods for assembly of the recombinant vectors are well-known. See, forexample, WO 00/15822 and other references cited therein, all of whichare incorporated by reference.

There are advantages and disadvantages to the various viral vectorsystems. The limits of how much DNA can be packaged is one determinantin choosing which system to employ. rAAV tend to be limited to about 4.5kb of DNA, whereas lentivirus (e.g., retrovirus) system can accommodate4-5 kb.

AAV Vectors

Adeno-associated viruses are small, single-stranded DNA viruses whichrequire a helper virus for efficient replication (Berns, K I,Parvoviridae: the viruses and their replication, p. 1007-1041 (vol. 2),in Fields, B N et al., Fundamental Virology, 3rd Ed., (Lippincott-RavenPublishers, Philadelphia (1995)). The 4.7 kb genome of AAV has twoinverted terminal repeats (ITR) and two open reading frames (ORFs) whichencode the Rep proteins and Cap proteins, respectively. The Rep readingframe encodes four proteins of molecular weights 78, 68, 52 and 40 kDa.These proteins primarily function in regulating AAV replication andrescue and integration of the AAV into the host cell chromosomes. TheCap reading frame encodes three structural proteins of molecular weights85 (VP 1), 72 (VP2) and 61 (VP3) kDa which form the virion capsid(Berns, supra). VP3 comprises >80% of total AAV virion proteins.

Flanking the rep and cap ORFs at the 5′ and 3′ ends are 145 bp ITRs, thefirst 125 bp's of which can form Y- or T-shaped duplex structures. Thetwo ITRs are the only cis elements essential for AAV replication,rescue, packaging and integration of the genome. Two conformations ofAAV ITRs called “flip” and “flop” exist (Snyder, R O et al., 1993, JVirol., 67:6096-6104; Berns, K I, 1990 Microbiol Rev, 54:316-29). Theentire rep and cap domains can be excised and replaced with a transgenesuch as a reporter or therapeutic transgene (Carter, B J, in Handbook ofParvoviruses, P. Tijsser, ed., CRC Press, pp. 155-168 (1990)).

AAVs have been found in many animal species, including primates, canine,fowl and human (Murphy, F A et al., The Classification and Nomenclatureof Viruses: Sixth Rept of the Int'l Comme on Taxonomy of Viruses, ArchVirol, Springer-Verlag, 1995). Six primate serotypes are known (AAV1,AAV2, AAV3, AAV4, AAV5 and AAV6).

The AAV ITR sequences and other AAV sequences employed in generating theminigenes, vectors, and capsids, and other constructs used in thepresent invention may be obtained from a variety of sources. Forexample, the sequences may be provided by any of the above 6 AAVserotypes or other AAV serotypes or other densoviruses, including bothpresently known human AAV and yet to yet-to-be-identified serotypes.Similarly, AAVs known to infect other animal species may be the sourceof ITRs used in the present molecules and constructs. Capsids from avariety of serotypes of AAV may be combined in various mixtures with theother vector components (e.g., WO01/83692 (Nov. 8, 2001) incorporated byreference). Many of these viral strains or serotypes are available fromthe American Type Culture Collection (ATCC), Manassas, Va., or areavailable from a variety of other sources (academic or commercial).

It may be desirable to synthesize sequences used in preparing thevectors and viruses of the invention using known techniques, based onpublished AAV sequences, e.g. available from a variety of databases. Thesource of the sequences utilized to prepare the present constructs isnot considered to be limiting. Similarly, the selection of the AAVserotype and species (of origin) is within the skill of the art and isnot considered limiting

The Minigene

As used herein, the AAV sequences are typically in the form of a rAAVconstruct (e.g., a minigene or cassette) which is packaged into a rAAVvirion. At minimum, the rAAV minigene is formed by AAV ITRs and aheterologous nucleic acid molecule for delivery to a host cell. Mostsuitably, the minigene comprises ITRs located 5′ and 3′ to theheterologous sequence. However, minigene comprising 5′ ITR and 3′ ITRsequences arranged in tandem, e.g. 5′ to 3′ or a head-to-tail, or inanother configuration may also be desirable. Other embodiments include aminigene with multiple copies of the ITRs, or one in which 5′ ITRs (orconversely, 3′ ITRs) are located both 5′ and 3′ to the heterologoussequence. The ITRs sequences may be located immediately upstream and/ordownstream of the heterologous sequence; intervening sequences may bepresent. The ITRs may be from AAV5, or from any other AAV serotype. Aminigene may include 5′ ITRs from one serotype and 3′ ITRs from another.

The AAV sequences used are preferably the 145 bp cis-acting 5′ and 3′ITR sequences (e.g., Carter, B J, supra). Preferably, the entire ITRsequence is used, although minor modifications are permissible. Methodsfor modifying these ITR sequences are well-known (e.g., Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, 3^(rd) Edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 2001; Brent, R et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 2003;Ausubel, F M et al., eds., Short Protocols in Molecular Biology, 5^(th)edition, Current Protocols, 2002; Carter et al., supra; and Fisher, K etal., 1996 J Virol. 70:520-32). It is conventional to engineer the rAAVvirus using known methods (e.g., Bennett, J et al. 1999, supra). Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the heterologous sequence, preferablythe Chop2 sequence, flanked by the 5′ and 3′ AAV ITR sequences.

The heterologous sequence encodes a protein or polypeptide which isdesired to be delivered to and expressed in a cell. The presentinvention is directed to Chop2 sequences under the control of a selectedpromoter and other conventional vector regulatory components.

The Transgene Being Targeted and Expressed

In a most preferred embodiment, the heterologous sequence is a nucleicacid molecule that functions as a transgene. The term “transgene” asused herein refers to a nucleic acid sequence heterologous to the AAVsequence, and encoding a desired product, preferably Chop2 and theregulatory sequences which direct or modulate transcription and/ortranslation of this nucleic acid in a host cell, enabling expression insuch cells of the encoded product. Preferred polypeptide products arethose that can be delivered to the eye, particularly to retinal neurons.

The transgene is delivered and expressed in order to treat or otherwiseimprove the vision status of a subject with an ocular disorder that mayresult from any of a number of causes, including mutations in a normalphotoreceptor-specific gene. The targeted ocular cells may bephotoreceptor cells (if not totally degenerated) or, more preferably,other retinal neurons, namely, bipolar cells and retinal ganglion cells.

Using an mGluR6 promoter operatively linked to a Chop2 opsin codingsequence and a reporter gene, e.g. GFP or another fluorescent protein,an insert of about 4.5 kb is preferred—1 kb for the opsin, 0.7 kb forthe reporter, 10 kb—for the mGluR6 promoter region and about 0.4 kb forconventional transcriptional regulatory factors.

Use of different opsin genes allows selection of desired wavelengths asthe absorption maxima of different channel proteins differ. In oneembodiment, the reported gene is moved to the red part of the visualspectrum.

Similarly, based on the studies reported herein, the brightness of thelight needed to stimulate evoked potential in transduced mouse retinas,indicates that a channel opsin with increased light sensitivity may bemore desirable. This can be achieved by selection of a suitablenaturally occurring opsin, for example other microbial-type rhodopsins,or by modifying the light sensitivity of Chop2 as well as its otherproperties, such as ion selectivity and spectral sensitivity, to producediversified light-sensitive channels to better fit the need for visionrestoration.

Different transgenes may be used to encode separate subunits of aprotein being delivered, or to encode different polypeptides theco-expression of which is desired. If a single transgene includes DNAencoding each of several subunits, the DNA encoding each subunit may beseparated by an internal ribozyme entry site (IRES), which is preferredfor short subunit-encoding DNA sequences (e.g., total DNA, includingIRES is <5 kB). Other methods which do not employ an IRES may be usedfor co-expression, e.g. the use of a second internal promoter, analternative splice signal, a co- or post-translational proteolyticcleavage strategy, etc., all of which are known in the art.

The coding sequence or non-coding sequence of the nucleic acids usefulherein preferably are codon-optimized for the species in which they areto be expressed. Such codon-optimization is routine in the art.

While a preferred transgene encodes a full length polypeptide,preferably Chop2 (SEQ ID NO:6, the present invention is also directed tovectors that encode a biologically active fragment or a conservativeamino acid substitution variant of Chop2 (or of any aother polypeptideof the invention to be expressed in retinal neurons). Non-limitingexamples of useful fragments are the polypeptide with the sequence SEQID NO:3 and SEQ ID NO:8. The fragment or variant is expressed by thetargets cells being transformed and is able to endow such cells withlight sensitivity that is functionally equivalent to that of the fulllength or substantially full length polypeptide having a native, ratherthan variant, amino acid sequence. A biologically active fragment orvariant is a “functional equivalent”—a term that is well understood inthe art and is further defined in detail herein. The requisitebiological activity of the fragment or variant, using any methoddisclosed herein or known in the art to establish activity of a channelopsin, has the following activity relative to the wild-type nativepolypeptide: about 50%, about 55%, about 60 %, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, andany range derivable therein, such as, for example, from about 70% toabout 80%, and more preferably from about 81% to about 90%; or even morepreferably, from about 91% to about 99%.

It should be appreciated that any variations in the coding sequences ofthe present nucleic acids and vectors that, as a result of thedegeneracy of the genetic code, express a polypeptide of the samesequence, are included within the scope of this invention.

The amino acid sequence identity of the variants of the presentinvention are determined using standard methods, typically based oncertain mathematical algorithms. In a preferred embodiment, the percentidentity between two amino acid sequences is determined using theNeedleman and Wunsch (J. Mol. Biol. 48:444-453 (1970) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at http://www.gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of Meyers andMiller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. The nucleotide and aminoacid sequences of the present invention can further be used as a “querysequence” to perform a search against public databases, for example, toidentify other family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (Altschul et al. (1990)J. Mol. Biol. 215:403-10). BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to, e.g. DAN encoding Chop2 of C. reinhardtii.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences homologous to theappropriate reference protein such as Chop2. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized (Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See World Wide Web URL ncbi.nlm.nih.gov.

The preferred amino acid sequence variant has the following degrees ofsequence identity with the native, full length channel opsinpolypeptide, preferably Chop2 from C. reinhardtii (SEQ ID NO:6) or witha fragment thereof (e.g., SEQ ID NO:3 or 8): about 50%, about 55%, abou60 %, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and anyrange derivable therein, such as, for example, from about 70% to about80%, and more preferably from about 81% to about 90%; or even morepreferably, from about 91% to about 99% identity. A preferredbiologically active fragment comprises or consists of SEQ ID NO:3, whichcorresponds to residues 1-315 of SEQ ID NO:6, or comprises or consistsof SEQ ID NO:8.

Any of a number of known recombinant methods are used to produce a DNAmolecule encoding the fragment or variant. For production of a variant,it is routine to introduce mutations into the coding sequence togenerate desired amino acid sequence variants of the invention.Site-directed mutagenesis is a well-known technique for which protocolsand reagents are commercially available (e.g., Zoller, M J et al., 1982,Nucl Acids Res 10:6487-6500; Adelman, J P et al., 1983, DNA 2:183-93).These mutations include simple deletions or insertions, systematicdeletions, insertions or substitutions of clusters of bases orsubstitutions of single bases.

In terms of functional equivalents, it is well understood by thoseskilled in the art that, inherent in the definition of a “biologicallyfunctional equivalent” protein, polypeptide, gene or nucleic acid, isthe concept that there is a limit to the number of changes that may bemade within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent peptides are thus defined herein asthose peptides in which certain, not most or all, of the amino acids maybe substituted.

In particular, the shorter the length of the polypeptide, the feweramino acids changes should be made. Longer fragments may have anintermediate number of changes. The full length polypeptide protein willhave the most tolerance for a larger number of changes. It is also wellunderstood that where certain residues are shown to be particularlyimportant to the biological or structural properties of a polypeptideresidues in a binding regions or an active site, such residues may notgenerally be exchanged. In this manner, functional equivalents aredefined herein as those poly peptides which maintain a substantialamount of their native biological activity.

For a detailed description of protein chemistry and structure, seeSchulz, G E et al., Principles of Protein Structure, Springer-Verlag,New York, 1978, and Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. The types of substitutions that may be madein the protein molecule may be based on analysis of the frequencies ofamino acid changes between a homologous protein of different species,such as those presented in Table 1-2 of Schulz et al. (supra) and FIG.3-9 of Creighton (supra). Based on such an analysis, conservativesubstitutions are defined herein as exchanges within one of thefollowing five groups:

1 Small aliphatic, nonpolar or slightly Ala, Ser, Thr (Pro, Gly); polarresidues 2 Polar, negatively charged residues and Asp, Asn, Glu, Gln;their amides 3 Polar, positively charged residues His, Arg, Lys; 4 Largealiphatic, nonpolar residues Met, Leu, Ile, Val (Cys) 5 Large aromaticresidues Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking a side chain andthus imparts flexibility to the chain. Pro, because of its unusualgeometry, tightly constrains the chain. Cys can participate in disulfidebond formation, which is important in protein folding.

The hydropathy index of amino acids may also be considered in selectingvariants. Each amino acid has been assigned a hydropathy index on thebasis of their hydrophobicity and charge characteristics, these are: Ile(+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala(+1.8); Glycine (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3);Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5);Lys (−3.9); and Arg (−4.5). The importance of the hydropathy index inconferring interactive biological function on a proteinaceous moleculeis generally understood in the art (Kyte and Doolittle, 1982, J. Mol.Biol. 157:105-32). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathy index orscore and still retain a similar biological activity. In making changesbased upon the hydropathy index, the substitution of amino acids whosehydropathy indices are within ±2 is preferred, those which are within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred. It is also understood in the art that thesubstitution of like amino acids can be made effectively on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide thereby created is intended for use in certain of thepresent embodiments. U.S. Pat. No. 4,554,101, discloses that thegreatest local average hydrophilicity of a proteinaceous molecule, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the molecule. See U.S. Pat. No. 4,554,101for a hydrophilicity values. In making changes based upon similarhydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

Regulatory Sequences

The minigene or transgene of the present invention includes appropriatesequences operably linked to the coding sequence or ORF to promote itsexpression in a targeted host cell. “Operably linked” sequences includeboth expression control sequences such as. promoters that are contiguouswith the coding sequences and expression control sequences that act intrans or distally to control the expression of the polypeptide product.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., Kozak consensus sequence); sequences thatenhance nucleic acid or protein stability; and when desired, sequencesthat enhance protein processing and/or secretion. Many varied expressioncontrol sequences, including native and non-native, constitutive,inducible and/or tissue-specific, are known in the art and may beutilized herein. depending upon the type of expression desired.

Expression control sequences for eukaryotic cells typically include apromoter, an enhancer, such as one derived from an immunoglobulin gene,SV40, CMV, etc., and a polyadenylation sequence which may include splicedonor and acceptor sites. The polyadenylation sequence generally isinserted 3′ to the coding sequence and 5′ to the 3′ ITR sequence. PolyAfrom bovine growth hormone is a suitable sequence.

The regulatory sequences useful herein may also contain an intron, suchas one located between the promoter/enhancer sequence and the codingsequence. One useful intron sequence is derived from SV40, and isreferred to as the SV40 T intron sequence. Another includes thewoodchuck hepatitis virus post-transcriptional element. (See, forexample, Wang L and Verma, I, 1999, Proc Nat'l Acad Sci USA,96:3906-10).

An IRES sequence, or other suitable system as discussed above, may beused to produce more than one polypeptide from a single transcript. nexemplary IRES is the poliovirus IRES which supports transgeneexpression in photoreceptors, RPE and ganglion cells. Preferably, theIRES is located 3′ to the coding sequence in the rAAV vector.

The promoter may be selected from a number of constitutive or induciblepromoters that can drive expression of the selected transgene in anocular setting, preferably in retinal neurons. A preferred promoter is“cell-specific”, meaning that it is selected to direct expression of theselected transgene in a particular ocular cell type, such asphotoreceptor cells.

Examples of useful constitutive promoters include the exemplified??? CMVimmediate early enhancer/chicken β-actin (CβA) promoter-exon 1-intron 1element, the RSV LTR promoter/enhancer, the SV40 promoter, the CMVpromoter, the dihydrofolate reductase (DHFR) promoter, and thephosphoglycerol kinase (PGK) promoter.

Additional useful promoters are disclosed in W. W. Hauswirth et al.,1998, W098/48027 and A. M. Timmers et al., 2000, WO00/15822. Promotersthat were found to drive RPE cell-specific gene expression in vivoinclude (1) a 528-bp promoter region (bases 1-528 of a murine 11-cisretinol dehydrogenase (RDH) gene (Driessen, C A et al., 1995, Invest.Ophthalmo!. Vis. Sci. 36:1988-96; Simon, A. et al., 1995, J. Biol. Chem270:1107-12, 1995; Simon, A. et al., 1996, Genomics 36:424-3) GenbankAccession Number X97752); (2) a 2274-bp promoter region) from a humancellular retinaldehyde-binding protein (CRALBP) gene (Intres, R et al.,1994, J. Bio!. Chem. 269:25411-18; Kennedy, B N et al., 1998, J. Bio!.Chem. 273:5591-8, 1998), Genbank Accession Number L34219); and (3) a1485-bp promoter region from human RPE65 (Nicoletti, A et al., 1998,Invest. Ophthalmol. Vis. Sci. 39:637-44, Genbank Accession NumberU20510). These three promoters (labeled with the following SEQ IDnumbers in WO00/15822″ 2.3 amd 3) promoted RPE-cell specific expressionof GFP. It is envisioned that minor sequence variations in the variouspromoters and promoter regions discussed herein—whether additions,deletions or mutations, whether naturally occurring or introduced invitro, will not affect their ability to drive expression in the cellulartargets of the present invention. Furthermore, the use of otherpromoters, even if not yet discovered, that are characterized byabundant and/or specific expression in retinal cells, particularly inbipolar or ganglion cells, is specifically included within the scope ofthis invention.

An inducible promoter is used to control the amount and timing ofproduction of the transgene product in an ocular cell. Such promoterscan be useful if the gene product has some undesired, e.g. toxic,effects in the cell if it accumulates excessively. Inducible promotersinclude those known in the art, such as the Zn-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (M MTV) promoter; the T7 promoter; the ecdysoneinsect promoter; the tetracycline-repressible system; thetetracycline-inducible system; the RU486-inducible system; and therapamycin-inducible system. Any inducible promoter the action of whichis tightly regulated and is specific for the particular target ocularcell type, may be used. Other useful types of inducible promoters areones regulated by a specific physiological state, e.g. temperature,acute phase, a cell's replicating or differentiation state.

Selection of the various vector and regulatory elements for use hereinare conventional, well-described, and readily available. See, e.g.Sambrook et al., supra; and Ausubel et al., supra. It will be readilyappreciated that not all vectors and expression control sequences willfunction equally well to express the present transgene, preferablyChop2. Clearly, the skilled artisan may apply routine selection amongthe known expression control sequences without departing from the scopeof this invention and based upon general knowledge as well as theguidance provided herein. One skilled in the art can select one or moreexpression control sequences, operably link them to the coding sequencebeing expressed to make a minigene, insert the minigene or vector intoan AAV vector, and cause packaging of the vector into infectiousparticles or virions following one of the known packaging methods forrAAV.

Production of the rAAV

The rAAV used in the present invention may be constructed and producedusing the materials and methods described herein and those well-known inthe art. The methods that are preferred for producing any construct ofthis invention are conventional and include genetic engineering,recombinant engineering, and synthetic techniques, such as those setforth in reference cited above.

Briefly, to package an rAAV construct into an rAAV virion, a sequencesnecessary to express AAV rep and AAV cap or functional fragments thereofas well as helper genes essential for AAV production must be present inthe host cells. See, for example U.S. Patent Pub. 2007/0015238, whichdescribes production of pseudotyped rAAV virion vectors encoding AAV Repand Cap proteins of different serotypes and AdV transcription productsthat provide helper functions For example, AAV rep and cap sequences maybe introduced into the host cell in any known manner including, withoutlimitation, transfection, electroporation, liposome delivery, membranefusion, biolistic deliver of DNA-coated pellets, viral infection andprotoplast fusion. Devices specifically adapted for delivering DNA tospecific regions within and around the eye for the purpose of genetherapy have been described recently (for example, U.S. Patent Pub.2005/0277868, incorporated by reference) are used within the scope ofthis invention. Such devices utilize electroporation andelectromigration, providing, e.g. two electrodes on a flexible supportthat can be placed behind the retina. A third electrode is part of ahollow support, which can also be used to inject the molecule to thedesired area. The electrodes can be positioned around the eye, includingbehind the retina or within the vitreous.

These sequences may exist stably in the cell as an episome or be stablyintegrated into the cell's genome. They may also be expressed moretransiently in the host cell. As an example, a useful nucleic acidmolecule comprises, from 5′ to 3′, a promoter, an optional spacerbetween the promoter and the start site of the rep sequence, an AAV repsequence, and an AAV cap sequence.

The rep and cap sequences, along with their expression controlsequences, are preferably provided in a single vector, though they maybe provided separately in individual vectors. The promoter may be anysuitable constitutive, inducible or native promoter. The deliverymolecule that provides the Rep and Cap proteins may be in any form.,preferably a plasmid which may contain other non-viral sequences, suchas those to be employed as markers. This molecule typically excludes theAAV ITRs and packaging sequences. To avoid the occurrence of homologousrecombination, other viral sequences, particularly adenoviral sequences,are avoided. This plasmid is preferably one that is stably expressed.

Conventional genetic engineering or recombinant DNA techniques describedin the cited references are used. The rAAV may be produced using atriple transfection method with either the calcium phosphate (Clontech)or Effectene™ reagent (Qiagen) according to manufacturer's instructions.See, also, Herzog et al., 1999, Nat. Med. 5:56-63.

The rAAV virions are produced by culturing host cells comprising a rAAVas described herein which includes a rAAV construct to be packaged intoa rAAV virion, an AAV rep sequence and an AAV cap sequence, all undercontrol of regulatory sequences directing expression.

Suitable viral helper genes, such as adenovirus E2A, E4Orf6 and VA, maybe added to the culture preferably on separate plasmids. Thereafter, therAAV virion which directs expression of the transgene is isolated in theabsence of contaminating helper virus or wildtype AAV.

It is conventional to assess whether a particular expression controlsequence is suitable for a given transgene, and choose the one mostappropriate for expressing the transgene. For example, a target cell maybe infected in vitro, and the number of copies of the transgene in thecell monitored by Southern blots or quantitative PCR. The level of RNAexpression may be monitored by Northern blots quantitative RT-PCR. Thelevel of protein expression may be monitored by Western blot,immunohistochemistry, immunoassay including enzyme immunoassay (EIA)such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays(RIA) or by other methods. Specific embodiments are described in theExamples below.

Pharmaceutical Compositions and Methods of the Invention

The rAAV that comprises the Chop2 transgene and cell-specific promoterfor use in the target ocular cell as described above should be assessedfor contamination using conventional methods and formulated into asterile or aseptic pharmaceutical composition for administration by, forexample, subretinal injection.

Such formulations comprise a pharmaceutically and/or physiologicallyacceptable vehicle, diluent, carrier or excipient, such as bufferedsaline or other buffers, e.g. HEPES, to maintain physiologic pH. For adiscussion of such components and their formulation, see, generally,Gennaro, A E., Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins Publishers; 2003 or latest edition). Seealso, WO00/15822. If the preparation is to be stored for long periods,it may be frozen, for example, in the presence of glycerol.

The pharmaceutical composition described above is administered to asubject having a visual or blinding disease by any appropriate route,preferably by intravitreal or subretinal injection, depending on theretinal layer being targeted.

Disclosures from Bennett and colleagues (cited herein) concern targetingof retinal pigment epithelium—the most distal layer from the vitrealspace. According to the present invention, the DNA construct is targetedto either retinal ganglion cells or bipolar cells. The ganglion cellsare reasonably well-accessible to intravitreal injection as disclosedherein. Intravitreal and/or subretinal injection can provide thenecessary access to the bipolar cells, especially in circumstances inwhich the photoreceptor cell layer is absent due to degeneration—whichis the case in certain forms of degeneration that the present inventionis intended to overcome.

To test for the vector's ability to express the transgene, specificallyin mammalian retinal neurons, by AAV-mediated delivery, a combination ofa preferred promoter sequence linked to a reporter gene such as LacZ orGFP linked to a SV40 poly A sequence can be inserted into a plasmid andpackaged into rAAV virus particles, concentrated, tested forcontaminating adenovirus and titered for rAAV using an infectious centerassay. The right eyes of a number of test subjects, preferably inbredmice, are injected sub-retinally with about 1 μl of the rAAV preparation(e.g., greater than about 10¹⁰ infectious units ml). Two weeks later,the right (test) and left (control) eyes of half the animals areremoved, fixed and stained with an appropriate substrate or antibody orother substance to reveal the presence of the reporter gene. A majorityof the test retinas in injected eyes will exhibited a focal stainedregion, e.g. blue for LacZ/Xgal, or green for GFP consistent with asubretinal bleb of the injected virus creating a localized retinaldetachment. All control eyes are negative for the reporter gene product.Reporter gene expression examined in mice sacrificed at later periods isdetected for at least 10 weeks post-injection, which suggests persistentexpression of the reporter transgene.

An effective amount of rAAV virions carrying a nucleic acid sequenceencoding the Chop2 DNA under the control of the promoter of choice,preferably a constitutive CMV promoter or a cell-specific promoter suchas mGluR6, is preferably in the range of between about 10¹⁰ to about10¹³ rAAV infectious units in a volume of between about 150 and about800 μl per injection. The rAAV infectious units can be measuredaccording to McLaughlin, S K et al., 1988, J Virol 62:1963. Morepreferably, the effective amount is between about 10¹⁰ and about 10¹²rAAV infectious units and the injection volume is preferably betweenabout 250 and about 500 μl. Other dosages and volumes, preferably withinthese ranges but possibly outside them, may be selected by the treatingprofessional, taking into account the physical state of the subject(preferably a human), who is being treated, including, age, weight,general health, and the nature and severity of the particular oculardisorder.

It may also be desirable to administer additional doses (“boosters”) ofthe present nucleic acid or rAAV compositions. For example, dependingupon the duration of the transgene expression within the ocular targetcell, a second treatment may be administered after 6 months or yearly,and may be similarly repeated. Neutralizing antibodies to AAV are notexpected to be generated in view of the routes and doses used, therebypermitting repeat treatment rounds.

The need for such additional doses can be monitored by the treatingprofessional using, for example, well-known electrophysiological andother retinal and visual function tests and visual behavior tests. Thetreating professional will be able to select the appropriate testsapplying routine skill in the art. It may be desirable to inject largervolumes of the composition in either single or multiple doses to furtherimprove the relevant outcome parameters.

Restoration or Improvement of Light Sensitivity and Vision

Both in vitro and in vivo studies to assess the various parameters ofthe present invention may be used, including recognized animal models ofblinding human ocular disorders. Large animal models of humanretinopathy, e.g. childhood blindness, are useful. The examples providedherein allow one of skill in the art to readily anticipate that thismethod may be similarly used in treating a range of retinal diseases.

While earlier studies by others have demonstrated that retinaldegeneration can be retarded by gene therapy techniques, the presentinvention demonstrates a definite physiological recovery of function,which is expected to generate or improve various parameters of vision,including behavioral parameters.

Behavioral measures can be obtained using known animal models and tests,for example performance in a water maze, wherein a subject in whomvision has been preserved or restored to varying extents will swimtoward light (Hayes, J M et al., 1993, Behav Genet 23:395-403).

In models in which blindness is induced during adult life or congenitalblindness develops slowly enough that the individual experiences visionbefore losing it, training of the subject in various tests may be done.In this way, when these tests are re-administered after visual loss totest the efficacy of the present compositions and methods for theirvision-restorative effects, animals do not have to learn the tasks denovo while in a blind state. Other behavioral tests do not requirelearning and rely on the instinctiveness of certain behaviors. Anexample is the optokinetic nystagmus test (Balkema G W et al., 1984,Invest Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et al., 1976,Vision Res. 16:1169-71).

As is exemplified herein, the transfection of retinal neurons with DNAencoding Chop2 provides residual retinal neurons, principally bipolarcells and ganglion cells, with photosensitive membrane channels. Thus,it was possible to measure, with a strong light stimulus, thetransmission of a visual stimulus to the animal's visual cortex, thearea of the brain responsible for processing visual signals; thistherefore constitutes a form of vision, as intended herein. Such visionmay differ from forms of normal human vision and may be referred to as asensation of light, also termed “light detection” or “light perception.”

Thus, the term “vision” as used herein is defined as the ability of anorganism to usefully detect light as a stimulus for differentiation oraction. Vision is intended to encompass the following:

-   1. Light detection or perception—the ability to discern whether or    not light is present-   2. Light projection—the ability to discern the direction from which    a light stimulus is coming;-   3. Resolution—the ability to detect differing brightness levels    (i.e., contrast) in a grating or letter target; and-   4. Recognition—the ability to recognize the shape of a visual target    by reference to the differing contrast levels within the target.

Thus, “vision” includes the ability to simply detect the presence oflight. This opens the possibility to train an affected subject who hasbeen treated according to this invention to detect light, enabling theindividual to respond remotely to his environment however crude thatinteraction might be. In one example, a signal array is produced towhich a low vision person can respond to that would enhance the person'sability to communicate by electronic means remotely or to performeveryday tasks. In addition such a person's mobility would bedramatically enhanced if trained to use such a renewed sense of lightresulting from “light detection.” The complete absence of lightperception leaves a person with no means (aside from hearing and smell)to discern anything about objects remote to himself.

The methods of the present invention that result in light perception,even without full normal vision, also improve or permit normallyregulated circadian rhythms which control many physiological processesincluding sleep-wake cycles and associated hormones. Although some blindindividuals with residual retinal ganglion cells (RGCs) can mediatetheir rhythms using RGC melanopsin, it is rare for them to do so. Thus,most blind persons have free-running circadian rhythms. Even when suchindividuals do utilize the melanopsin pathway, the effect is very weakeffect. The methods of the present invention are thus expected toimprove health status of blind individuals by enabling absent lightentrainment or improving weakened (melanopsin-mediated) lightentrainment of their circadian rhythms. This leads to better health andwell-being of these subjects.

In addition to circadian rhythms, the present invention provides a basisto improve deficits in other light-induced physiological phenomena.Photoreceptor degeneration may result in varying degrees of negativemasking, or suppression, of locomotor activity during the intervals inthe circadian cycle in which the individual should be sleeping. Anotherresult is suppression of pineal melatonin. Both of these contribute tothe entrainment process. Thus, improvement in these responses oractivities in a subject in whom photoreceptors are degenerating or havedegenerated contributes, independently of vision per se, to appropriatesleep/wake cycles that correspond with the subject's environment in thereal world.

Yet another benefit of the present invention is normalization ofpupillary light reflexes because regulation of pupil size helps modulatethe effectiveness of light stimuli in a natural feed back loop. Thus,the present invention promotes re-establishment of this natural feedbackloop, making vision more effective in subject treated as describedherein.

In certain embodiments, the present methods include the measurement ofvision before, and preferably after, administering a vector comprising,for example, DNA encoding Chop2. Vision is measured using any of anumber of methods well-known in the art or ones not yet establshed. Mostpreferred herein are the following visual responses:

-   (1) A light detection response by the subject after exposure to a    light stimulus—in which evdence is sought for a reliable response of    an indication or movement in the general direction of the light by    the subject individual when the light it is turned on is .-   (2) a light projection response by the subject after exposure to a    light stimulus in which evidence is sought for a reliable response    of indication or movement in the specific direction of the light by    the individual when the light is turned on.-   (3) light resolution by the subject of a light vs. dark patterned    visual stimulus, which measures the subject's capability of    resolving light vs dark patterned visual stimuli as evidenced by:    -   (a) the presence of demonstrable reliable optokinetically        produced nystagmoid eye movements and/or related head or body        movements that demonstrate tracking of the target (see above)        and/or    -   (b). the presence of a reliable ability to discriminate a        pattern visual stimulus and to indicate such discrimination by        verbal or non-verbal means, including, for example pointing, or        pressing a bar or a button; or-   (4) electrical recording of a visual cortex response to a light    flash stimulus or a pattern visual stimulus, which is an endpoint of    electrical transmission from a restored retina to the visual cortex.    Measurement may be by electrical recording on the scalp surface at    the region of the visual cortex, on the cortical surface, and/or    recording within cells of the visual cortex.

It is known in the art that it is often difficult to make children whohave only light perception appreciate that they have this vision.Training is required to get such children to react to their visualsensations. Such a situation is mimicked in the animal studiesexemplified below. Promoting or enhancing light perception, which thecompositions and methods of the present invention will accomplish, isvaluable because patients with light perception not only are trainableto see light, but they can usually be trained to detect the visualdirection of the light, thus enabling them to be trained in mobility intheir environment. In addition, even basic light perception can be usedby visually impaired individuals, including those whose vision isimproved using the present compositions and methods, along withspecially engineered electronic and mechanical devices to enable theseindividuals to accomplish specific daily tasks. Beyond this anddepending on their condition, they may even be able to be trained inresolution tasks such as character recognition and even reading if theirimpairment permits. Thus it is expected that the present inventionenhances the vision of impaired subjects to such a level that byapplying additional training methods, these individuals will achieve theabove objectives.

Low sensitivity vision may emulate the condition of a person with anight blinding disorder, an example of which is Retinitis Pigmentosa(RP), who has difficulty adapting to light levels in his environment andwho might use light amplification devices such as supplemental lightingand/or night vision devices.

Thus, the visual recovery that has been described in the animal studiesdescribed below would, in human terms, place the person on the low endof vision function. Nevertheless, placement at such a level would be asignificant benefit because these individuals could be trained inmobility and potentially in low order resolution tasks which wouldprovide them with a greatly improved level of visual independencecompared to total blindness.

The mice studied in the present Examples were rendered completely devoidof photoreceptors; this is quite rare, even in the worst human diseases.The most similar human state is RP. In most cases of RP, central visionis retained till the very end. In contrast, in the studied mouse model,the mouse becomes completely blind shortly after birth.

Common disorders encountered in low vision are described by J. Tasca andE. A. Deglin in Chap. 6 of Essentials of Low Vision Practice, R. L.Brilliant, ed., Butterworth Heinemann Publ., 1999, which is incorporatedby reference in its entirety. There is reference to similar degenerativeconditions, but these references show form vision that is measurable asvisual acuity. Ganglion cell layers are not retained in all forms of RP,so the present approach will not work for such a disorder.

When applying the present methods to humans with severe cases of RP, itis expected that central vision would be maintained for a time at somelow level while the peripheral retina degenerated first. It is thisdegenerating retina that is the target for re-activation using thepresent invention. In essence, these individuals would be able to retainmobility vision as they approached blindness gradually.

Subjects with macular degeneration, characterized by photoreceptor losswithin the central “sweet spot” of vision (Macula Lutea), are expectedto benefit by treatment in accordance with the present invention, inwhich case the resolution capability of the recovered vision would beexpected to be higher due to the much higher neuronal density within thehuman macula.

While it is expected that bright illumination of daylight and artificiallighting that may be used by a visually impaired individual will sufficefor many visual activities that are performed with vision that hasrecovered as a result of the present treatments. It is also possiblethat light amplification devices may be used, as needed, to furtherenhance the affected person's visual sensitivity. The human visionsystem can operate over a 10 log unit range of luminance. On the otherhand, microbial type rhodopsins, such as ChR2, operate over up to a 3log unit range of luminance. In addition, the light conditions thepatient encounters could fall outside of the operating range of thelight sensor. To compensate for the various light conditions, a lightpre-amplification or attenuation device could be used to expand theoperation range of the light conditions. Such device would contain acamera, imaging processing system, and microdisplays, which can neassembled from currently available technologies, such as night visiongoggles and/or 3D adventure and entertainment system. (See, for examplethe following URL on the Worldwide web—emagin.com/.)

The present invention may be used in combination with other forms ofvision therapy known in the art. Chief among these is the use of visualprostheses, which include retinal implants, cortical implants, lateralgeniculate nucleus implants, or optic nerve implants. Thus, in additionto genetic modification of surviving retinal neurons using the presentmethods, the subject being treated may be provided with a visualprosthesis before, at the same time as, or after the molecular method isemployed.

The effectiveness of visual prosthetics can be improved with training ofthe individual, thus enhancing the potential impact of the Chop2transformation of patient cells as contemplated herein. An example of anapproach to training is found in US 2004/0236389 (Fink et al.),incorporated by reference. The training method may include providing anon-visual reference stimulus to a patient having a visual prosthesisbased on a reference image. The non-visual reference stimulus isintended to provide the patient with an expectation of the visual imagethat the prosthesis will induce. Examples of non-visual referencestimuli are a pinboard, Braille text, or a verbal communication. Thevisual prosthesis stimulates the patient's nerve cells, including thosecells whose responsiveness has been improved by expressing Chop2 asdisclosed herein, with a series of stimulus patterns attempting toinduce a visual perception that matches the patient's expectedperception derived from the non-visual reference stimulus. The patientprovides feedback to indicate which of the series of stimulus patternsinduces a perception that most closely resembles the expectedperception. The patient feedback is used as a “fitness function” (alsoreferred to as a cost function or an energy function). Subsequentstimuli provided to the patient through the visual prosthesis are based,at least in part, on the previous feedback of the patient as to whichstimulus pattern(s) induce the perception that best matches the expectedperception. The subsequent stimulus patterns may also be based, at leastin part, on a fitness function optimization algorithm, such as asimulated annealing algorithm or a genetic algorithm.

Thus, in certain embodiments of this invention, the method of improvingor restoring vision in a subject further comprises training of thatsubject, as discussed above. Preferred examples of training methods are:

-   -   (a) habituation training characterized by training the subject        to recognize (i) varying levels of light and/or pattern        stimulation, and/or (ii) environmental stimulation from a common        light source or object as would be understood by one skilled in        the art; and    -   (b) orientation and mobility training characterized by training        the subject to detect visually local objects and move among said        objects more effectively than without the training.        In fact, any visual stimulation techniques that are typically        used in the field of low vision rehabilitation are applicable        here.

The remodeling of inner retinal neurons triggered by photoreceptordegeneration has raised a concerns about retinal-based rescue strategiesafter the death of photoreceptors (Strettoi and Pignatelli 2000, ProcNatl Acad Sci USA. 97:11020-5; Jones, B W et al., 2003, J Comp Neurol464:1-16 ; Jones, B W and Marc, R E, 2005, Exp Eye Res. 81:123-37;Jones, B W et al., 2005, Clin Exp Optom. 88:282-91). Retinal remodelingis believed to result from deafferentation, the loss of afferent inputsfrom photoreceptors—in other words, the loss of light induced activitiesSo after death of rods and coned, there is no light evoked input toretinal bipolar cells and ganglion cells, and through them to highervisual centers. In response to the loss of such input, the retina andhigher visual network are triggered to undergo remodeling, in a wayseeking other forms of inputs. Said otherwise, the retina needs to beused to sense light in order to maintain its normal network, and withthe loss of light sensing, the network will deteriorate via a remodelingprocess. This process is not an immediate consequence of photoreceptordeath; rather it is a slow process, providing a reasonably long windowfor intervention.

Thus, an additional utility of restoring light sensitivity to innerretinal neurons in accordance with the present invention is theprevention or delay in the remodeling processes in the retina, and,possibly, in the higher centers. Such retinal remodeling may haveundesired consequences such as corruption of inner retinal network,primarily the connection between bipolar and retinal ganglion cells. Byintroducing the light-evoked activities in bipolar cells or ganglioncells, the present methods would prevent or diminish the remodeling dueto the lack of input; the present methods introduce this missing input(either starting from bipolar cells or ganglion cells), and therebystabilize the retinal and higher visual center network. Thus,independently of its direct effects on vision, the present inventionwould benefit other therapeutic approaches such as photoreceptortransplantation or device implants,.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Synopsis of Examples (references cited in the following sections mayappear in a list at the end). Methods

A Chop2-GFP chimera was made by linking a nucleic acid encoding greenfluorescent protein (GFP) (part of SEQ ID NO: 1 as shown below) to anucleic acid (SEQ ID NO:2) encoding an active fragment (SEQ ID NO:3) ofchannelopsin-2 (Chop2) such that an expressed protein has the GFP linkedto the C-terminus of the Chop2 region. Both these sequences constitutethe “transgene” as discussed above. The Chop2-GFP DNA was transfectedinto HEK293 cells under control of a CMV promoter.

A viral construct (SEQ ID NO: 1) was made by subcloning the Chop2-GFPinto an AAV-2 viral cassette containing a CAG promoter. A map of thisconstruct is shown in FIG. 7. The viral vectors were injected into theeye of newborn rats. The expression of Chop2-GFP was examined by GFPfluorescence in retinal whole-mounts or slice sections. The function ofthe Chop2-GFP was assessed by whole-cell patch clamp recordings.

Results

Bright GFP fluorescence was detected within 18-24 hrs in HEK cells afterthe transfection. The fluorescence was localized predominantly to theplasma membrane. The preserve of the function of the Chop2-GFP chimerawas confirmed by patch-clamp recordings. Substantial light-gatedcurrents were also observed in the Chop2-GFP-expressing HEK cellswithout adding the exogenous all-trans retinal, indicating that asignificant number of functional Chop2-GFP channels were formed in HEKcells using only endogenous precursor for the chromophore group. Threeto four weeks after the injection, GFP fluorescence was observed in theretinal neurons of the injected eyes. Bright GFP-fluorescence wasobserved in many ganglion cells and horizontal cells, some amacrinecells, and, occasionally, bipolar cells for at least 10 weeks followinginjection. The Chop2-GFP-expressing retinal neurons exhibited robustmembrane depolarization in response to light stimulation and did notrequire an exogenous source of all-trans retinal.

Thus, the inventors demonstrated that the selected AAV vector constructefficiently targeted retinal ganglion cells and effectively deliveredthe Chop2-GFP cDNA and expressed protein at high levels afterintravitreal injection in both normal and diseased retinas. Whenendogenous retinal was bound to the Chop2, it could be photoswitched,and neural activity could be evoked in retinas and at cortical levels.This was shown by several techniques-initially by in vitro patch-clamprecordings of individual dissociated RGCs, followed by multielectrodearray recordings of whole-mount retina preparations representative of alarge population of RGCs. Finally, in vivo cortical recordings from liveblind mice demonstrated that critical connections were functionallymaintained to higher visual centers.

Conclusion

The progressive in vitro and in vivo results show that ectopicexpression of Chop2 is a therapeutic strategy for restoring lightsensitivity to a “blind” retina. Functional expression of a directlylight-gated membrane channel, Chop2, was demonstrated in rat retinalneurons in vivo. Thus, expression of light-gated membrane channels insecond- or third-order retinal neurons is a useful strategy forrestoration of light perception after photoreceptor degeneration.

Example I Materials and Methods DNA and Viral Vector Constructions

The DNA fragment encoding the N-terminal fragment (Met¹-Lys³¹⁵) of Chop2(Nagel et al., 2003) was cloned into pBluescript vector (Stratagene)containing the last exon of a mouse protamine 1 gene containingpolyadenylation signal (mP1) and GFP cDNA inserted in frame at the 3′end of the Chop2 coding fragment to produce a Chop2-GFP fusion protein.The function of Chop2-GFP chimera was verified in transfected HEK293cells.

The viral expression construct rAAV2-C AG-Chop2-GFP-WPR E was made bysubcloning the Chop2-GFP fragment into an adeno-associated (serotype-2)viral expression cassette. The viral cassette comprised a hybrid CMVenhancer/chicken β-actin promoter (CAG), a woodchuck posttranscriptionalregulatory element (WPRE), and a bovine growth hormone (BGH)polyadenylation sequence. Viral vectors were packaged and affinitypurified (GeneDetect).

The vector map is shown in FIG. 7.

The nucleic acid sequence of this vector (SEQ ID NO: 1) is shown belowin annotated form (with the annotations as described):

-   -   ITR's (at both ends) (UPPER CASE underscore)    -   CAG promoter sequence (Lower case, bold, italic)    -   Kozak sequence (lower case double underscore)    -   Chop2 coding sequence (lower case, bold)    -   Green fluorescent protein coding sequence (lower case, bold        underscored)    -   WPRE (regulatory element): (UPPER CASE)    -   The BGH Poly A sequence is not marked.

The remaining sequence (all lower case), including between Chop2 andGFP, is vector sequence

The Chop2 coding sequence from the above vector is shown below as SEQ IDNO:2. Numbering indicates both nucleotide number and codon number. Theencoded polypeptide (SEQ ID NO:3) is also shown. Again, this is theN-terminal 315 residues of Chop2 polypeptide (SEQ ID NO:6).

atg gat tat gga ggc gcc ctg agt gcc gtt ggg cgc gag ctg cta ttt  48 M   D   Y   G   G   A   L   S   A   V   G   R   E   L   L   F  16 gtaacg aac cca gta gtc gtc aat ggc tct gta ctt gtg cct gag gac  96 V   T   N   P   V   V   V   N   G   S   V   L   V   P   E   D  32 cagtgt tac tgc gcg ggc tgg att gag tcg cgt ggc aca aac ggt gcc 144 Q   C   Y   C   A   G   W   I   E   S   R   G   T   N   G   A  48 caaacg gcg tcg aac gtg ctg caa tgg ctt gct gct ggc ttc tcc atc 192 Q   T   A   S   N   V   L   Q   W   L   A   A   G   F   S   I  64 ctactg ctt atg ttt tac gcc tac caa aca tgg aag tca acc tgc ggc 240 L   L   L   M   F   Y   A   Y   Q   T   W   K   S   T   C   G  80 tgggag gag atc tat gtg tgc gct atc gag atg gtc aag gtg att ctt 288 W   E   E   I   Y   V   C   A   I   E   M   V   K   V   I   L  96 gagttc ttc ttc gag ttt aag aac ccg tcc atg ctg tat cta gcc aca 336 E   F   F   F   E   F   K   N   P   S   M   L   Y   L   A   T 112 ggccac cgc gtc cag tgg ttg cgt tac gcc gag tgg ctt ctc acc tgc 384 G   H   R   V   Q   W   L   R   Y   A   E   W   L   L   T   C 128 ccggtc att ctc att cac ctg tca aac ctg acg ggc ttg tcc aac gac 432 P   V   I   L   I   H   L   S   N   L   T   G   L   S   N   D 144 tacagc agg cgc act atg ggt ctg ctt gtg tct gat att ggc aca att 480 Y   S   R   R   T   M   G   L   L   V   S   D   I   G   T   I 160 gtgtgg ggc gcc act tcc gct atg gcc acc gga tac gtc aag gtc atc 528 V   W   G   A   T   S   A   M   A   T   G   Y   V   K   V   I 176 ttcttc tgc ctg ggt ctg tgt tat ggt gct aac acg ttc ttt cac gct 576 F   F   C   L   G   L   C   Y   G   A   N   T   F   F   H   A 192 gccaag gcc tac atc gag ggt tac cat acc gtg ccg aag ggc cgg tgt 624 A   K   A   Y   I   E   G   Y   H   T   V   P   K   G   R   C 208 cgccag gtg gtg act ggc atg gct tgg ctc ttc ttc gta tca tgg ggt 672 R   Q   V   V   T   G   M   A   W   L   F   F   V   S   W   G 224 atgttc ccc atc ctg ttc atc ctc ggc ccc gag ggc ttc ggc gtc ctg 720 M   F   P   I   L   F   I   L   G   P   E   G   F   G   V   L 240 agcgtg tac ggc tcc acc gtc ggc cac acc atc att gac ctg atg tcg 768 S   V   Y   G   S   T   V   G   H   T   I   I   D   L   M   S 256 aagaac tgc tgg ggt ctg ctc ggc cac tac ctg cgc gtg ctg atc cac 816 K   N   C   W   G   L   L   G   H   Y   L   R   V   L   I   H 272 gagcat atc ctc atc cac ggc gac att cgc aag acc acc aaa ttg aac 864 E   H   I   L   I   H   G   D   I   R   K   T   T   K   L   N 288 attggt ggc act gag att gag gtc gag acg ctg gtg gag gac gag gcc 912 I   G   G   T   E   I   E   V   E   T   L   V   E   D   E   A 304 gaggct ggc gcg gtc aac aag ggc acc ggc aag 945 E   A   G   A   V   N   K   G   T   G   K 315

A native nucleic acid sequence that encodes the full length Chop2protein of C. reinhardtii (GenBank Accession #AF461397) has thefollowing nucleotide sequence (SEQ ID NO:4). Note that the codingsequence begins at the ATG codon beginning at nt 28.

   1 gcatctgtcg ccaagcaagc attaaac ATG  gattatggag gcgccctgag tgccgttggg  61 cgcgagctgc tatttgtaac gaacccagta gtcgtcaatg gctctgtact tgtgcctgag 121 gaccagtgtt actgcgcggg ctggattgag tcgcgtggca caaacggtgc ccaaacggcg 181 tcgaacgtgc tgcaatggct tgctgctggc ttctccatcc tactgcttat gttttacgcc 241 taccaaacat ggaagtcaac ctgcggctgg gaggagatct atgtgtgcgc tatcgagatg 301 gtcaaggtga ttctcgagtt cttcttcgag tttaagaacc cgtccatgct gtatctagcc 361 acaggccacc gcgtccagtg gttgcgttac gccgagtggc ttctcacctg cccggtcatt 421 ctcattcacc tgtcaaacct gacgggcttg tccaacgact acagcaggcg caccatgggt 481 ctgcttgtgt ctgatattgg cacaattgtg tggggcgcca cttccgccat ggccaccgga 541 tacgtcaagg tcatcttctt ctgcctgggt ctgtgttatg gtgctaacac gttctttcac 601 gctgccaagg cctacatcga gggttaccac accgtgccga agggccggtg tcgccaggtg 661 gtgactggca tggcttggct cttcttcgta tcatggggta tgttccccat cctgttcatc 721 ctcggccccg agggcttcgg cgtcctgagc gtgtacggct ccaccgtcgg ccacaccatc 781 attgacctga tgtcgaagaa ctgctggggt ctgctcggcc actacctgcg cgtgctgatc 841 cacgagcata tcctcatcca cggcgacatt cgcaagacca ccaaattgaa cattggtggc 901 actgagattg aggtcgagac gctggtggag gacgaggccg aggctggcgc ggtcaacaag 961 ggcaccggca agtacgcctc ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag1021 aagggcattg acgtgcgcgc ctctctggac aacagcaagg aggtggagca ggagcaggcc1081 gccagggctg ccatgatgat gatgaacggc aatggcatgg gtatgggaat gggaatgaac1141 ggcatgaacg gaatgggcgg tatgaacggg atggctggcg gcgccaagcc cggcctggag1201 ctcactccgc agctacagcc cggccgcgtc atcctggcgg tgccggacat cagcatggtt1261 gacttcttcc gcgagcagtt tgctcagcta tcggtgacgt acgagctggt gccggccctg1321 ggcgctgaca acacactggc gctggttacg caggcgcaga acctgggcgg cgtggacttt1381 gtgttgattc accccgagtt cctgcgcgac cgctctagca ccagcatcct gagccgcctg1441 cgcggcgcgg gccagcgtgt ggctgcgttc ggctgggcgc agctggggcc catgcgtgac1501 ctgatcgagt ccgcaaacct ggacggctgg ctggagggcc cctcgttcgg acagggcatc1561 ctgccggccc acatcgttgc cctggtggcc aagatgcagc agatgcgcaa gatgcagcag1621 atgcagcaga ttggcatgat gaccggcggc atgaacggca tgggcggcgg tatgggcggc1681 ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg catgggcggc1741 ggcatgggca acggcatggg cggcaatggc atgaacggaa tgggtggcgg caacggcatg1801 aacaacatgg gcggcaacgg aatggccggc aacggaatgg gcggcggcat gggcggcaac1861 ggtatgggtg gctccatgaa cggcatgagc tccggcgtgg tggccaacgt gacgccctcc1921 gccgccggcg gcatgggcgg catgatgaac ggcggcatgg ctgcgcccca gtcgcccggc1981 atgaacggcg gccgcctggg taccaacccg ctcttcaacg ccgcgccctc accgctcagc2041 tcgcagctcg gtgccgaggc aggcatgggc agcatgggag gcatgggcgg aatgagcgga2101 atgggaggca tgggtggaat ggggggcatg ggcggcgccg gcgccgccac gacgcaggct2161 gcgggcggca acgcggaggc ggagatgctg cagaatctca tgaacgagat caatcgcctg2221 aagcgcgagc ttggcgag

The coding portion of SEQ ID NO:4 is shown below as SEQ ID NO:5,organized as 737 triplet codons (plus a stop codon) that encode a 737amino acid polypeptide. The ATG start codon and the TAA stop codon arehighlighted.

ATG  gat tat gga ggc gcc ctg agt gcc gtt ggg cgc gag ctg cta ttt gta acgaac cca gta gtc gtc aat ggc tct gta ctt gtg cct gag gac cag tgt tac tgcgcg ggc tgg att gag tcg cgt ggc aca aac ggt gcc caa acg gcg tcg aac gtgctg caa tgg ctt gct gct ggc ttc tcc atc cta ctg ctt atg ttt tac gcc taccaa aca tgg aag tca acc tgc ggc tgg gag gag atc tat gtg tgc gct atc gagatg gtc aag gtg att ctc gag ttc ttc ttc gag ttt aag aac ccg tcc atg ctgtat cta gcc aca ggc cac cgc gtc cag tgg ttg cgt tac gcc gag tgg ctt ctcacc tgc ccg gtc att ctc att cac ctg tca aac ctg acg ggc ttg tcc aac gactac agc agg cgc acc atg ggt ctg ctt gtg tct gat att ggc aca att gtg tggggc gcc act tcc gcc atg gcc acc gga tac gtc aag gtc atc ttc ttc tgc ctgggt ctg tgt tat ggt gct aac acg ttc ttt cac gct gcc aag gcc tac atc gagggt tac cac acc gtg ccg aag ggc cgg tgt cgc cag gtg gtg act ggc atg gcttgg ctc ttc ttc gta tca tgg ggt atg ttc ccc atc ctg ttc atc ctc ggc cccgag ggc ttc ggc gtc ctg agc gtg tac ggc tcc acc gtc ggc cac acc atc attgac ctg atg tcg aag aac tgc tgg ggt ctg ctc ggc cac tac ctg cgc gtg ctgatc cac gag cat atc ctc atc cac ggc gac att cgc aag acc acc aaa ttg aacatt ggt ggc act gag att gag gtc gag acg ctg gtg gag gac gag gcc gag gctggc gcg gtc aac aag ggc acc ggc aag tac gcc tcc cgc gag tcc ttc ctg gtcatg cgc gac aag atg aag gag aag ggc att gac gtg cgc gcc tct ctg gac aacagc aag gag gtg gag cag gag cag gcc gcc agg gct gcc atg atg atg atg aacggc aat ggc atg ggt atg gga atg gga atg aac ggc atg aac gga atg ggc ggtatg aac ggg atg gct ggc ggc gcc aag ccc ggc ctg gag ctc act ccg cag ctacag ccc ggc cgc gtc atc ctg gcg gtg ccg gac atc agc atg gtt gac ttc ttccgc gag cag ttt gct cag cta tcg gtg acg tac gag ctg gtg ccg gcc ctg ggcgct gac aac aca ctg gcg ctg gtt acg cag gcg cag aac ctg ggc ggc gtg gacttt gtg ttg att cac ccc gag ttc ctg cgc gac cgc tct agc acc agc atc ctgagc cgc ctg cgc ggc gcg ggc cag cgt gtg gct gcg ttc ggc tgg gcg cag ctgggg ccc atg cgt gac ctg atc gag tcc gca aac ctg gac ggc tgg ctg gag ggcccc tcg ttc gga cag ggc atc ctg ccg gcc cac atc gtt gcc ctg gtg gcc aagatg cag cag atg cgc aag atg cag cag atg cag cag att ggc atg atg acc ggcggc atg aac ggc atg ggc ggc ggt atg ggc ggc ggc atg aac ggc atg ggc ggcggc aac ggc atg aac aac atg ggc aac ggc atg ggc ggc ggc atg ggc aac ggcatg ggc ggc aat ggc atg aac gga atg ggt ggc ggc aac ggc atg aac aac atgggc ggc aac gga atg gcc ggc aac gga atg ggc ggc ggc atg ggc ggc aac ggtatg ggt ggc tcc atg aac ggc atg agc tcc ggc gtg gtg gcc aac gtg acg ccctcc gcc gcc ggc ggc atg ggc ggc atg atg aac ggc ggc atg gct gcg ccc cagtcg ccc ggc atg aac ggc ggc cgc ctg ggt acc aac ccg ctc ttc aac gcc gcgccc tca ccg ctc agc tcg cag ctc ggt gcc gag gca ggc atg ggc agc atg ggaggc atg ggc gga atg agc gga atg gga ggc atg ggt gga atg ggg ggc atg ggcggc gcc ggc gcc gcc acg acg cag gct gcg ggc ggc aac gcg gag gcg gag atgctg cag aat ctc atg aac gag atc aat cgc ctg aag cgc gag ctt ggc gag taa 2214 nt's

The full length Chop2 protein of C. reinhardtii (GenBank Accession#AF461397) encoded by SEQ ID NO's 3 and 4, has the following amino acidsequence, SEQ ID NO:6:

MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNG  50 AQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILE 100 FFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSR 150 RTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYI 200 EGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTV 250 GHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVE 300 TLVEDEAEAGAVNKGTGKYASRESFLVMRDKMKEKGIDVRASLDNSKEVE 350 QEQAARAAMMMMNGNGMGMGMGMNGMNGMGGMNGMAGGAKPGLELTPQLQ 400 PGRVILAVPDISMVDFFREQFAQLSVTYELVPALGADNTLALVTQAQNLG 450 GVDFVLIHPEFLRDRSSTSILSRLRGAGQRVAAFGWAQLGPMRDLIESAN 500 LDGWLEGPSFGQGILPAHIVALVAKMQQMRKMQQMQQIGMMTGGMNGMGG 550 GMGGGMNGMGGGNGMNNMGNGMGGGMGNGMGGNGMNGMGGGNGMNNMGGN 600 GMAGNGMGGGMGGNGMGGSMNGMSSGVVANVTPSAAGGMGGMMNGGMAAP 650 QSPGMNGGRLGTNPLFNAAPSPLSSQLGAEAGMGSMGGMGGMSGMGGMGG 700 MGGMGGAGAATTQAAGGNAEAEMLQNLMNEINRLKRELGE 737

Another useful Chop2 sequence useful for the present invention is anucleic acid of 933 nt's (including the stop codon) encoding a 310 aapolypeptide (a biologically active fragment of the full length nativeChop2) is a synthetic construct derived from Chlamydomonas reinhardtii″(See EF474017 and Zhang et al., 2007, Nature in press). This sequence iscodon-optimized for human expression. The nt sequence shown below is SEQID NO:7, and the encoded a.a. sequence shown is SEQ ID NO:8. Thepolypeptide with the a.a. sequence SEQ ID NO:8 is a fragment of SEQ IDNO:6 truncated at the C-terminus and with Pro replacing Asn at 310.

atg gac tat ggc ggc gct ttg tct gcc gtc gga cgc gaa ctt ttg ttc  48 M   D   Y   G   G   A   L   S   A   V   G   R   E   L   L   F  16 gttact aat cct gtg gtg gtg aac ggg tcc gtc ctg gtc cct gag gat  96 V   T   N   P   V   V   V   N   G   S   V   L   V   P   E   D  32 caatgt tac tgt gcc gga tgg att gaa tct cgc ggc acg aac ggc gct 144 Q   C   Y   C   A   G   W   I   E   S   R   G   T   N   G   A  48 cagacc gcg tca aat gtc ctg cag tgg ctt gca gca gga ttc agc att 192 Q   T   A   S   N   V   L   Q   W   L   A   A   G   F   S   I  64 ttgctg ctg atg ttc tat gcc tac caa acc tgg aaa tct aca tgc ggc 240 L   L   L   M   F   Y   A   Y   Q   T   W   K   S   T   C   G  80 tgggag gag atc tat gtg tgc gcc att gaa atg gtt aag gtg att ctc 288 W   E   E   I   Y   V   C   A   I   E   M   V   K   V   I   L  96 gagttc ttt ttt gag ttt aag aat ccc tct atg ctc tac ctt gcc aca 336 E   F   F   F   E   F   K   N   P   S   M   L   Y   L   A   T 112 ggacac cgg gtg cag tgg ctg cgc tat gca gag tgg ctg ctc act tgt 384 G   H   R   V   Q   W   L   R   Y   A   E   W   L   L   T   C 128 cctgtc atc ctt atc cac ctg agc aac ctc acc ggc ctg agc aac gac 432 P   V   I   L   I   H   L   S   N   L   T   G   L   S   N   D 144 tacagc agg aga acc atg gga ctc ctt gtc tca gac atc ggg act atc 480 Y   S   R   R   T   M   G   L   L   V   S   D   I   G   T   I 160 gtgtgg ggg gct acc agc gcc atg gca acc ggc tat gtt aaa gtc atc 528 V   W   G   A   T   S   A   M   A   T   G   Y   V   K   V   I 176 ttcttt tgt ctt gga ttg tgc tat ggc gcg aac aca ttt ttt cac gcc 576 F   F   C   L   G   L   C   Y   G   A   N   T   F   F   H   A 192 gccaaa gca tat atc gag ggt tat cat act gtg cca aag ggt cgg tgc 624 A   K   A   Y   I   E   G   Y   H   T   V   P   K   G   R   C 208 cgccag gtc gtg acc ggc atg gca tgg ctg ttt ttc gtg agc tgg ggt 672 R   Q   V   V   T   G   M   A   W   L   F   F   V   S   W   G 224 atgttc cca att ctc ttc att ttg ggg ccc gaa ggt ttt ggc gtc ctg 720 M   F   P   I   L   F   I   L   G   P   E   G   F   G   V   L 240 agcgtc tat ggc tcc acc gta ggt cac acg att att gat ctg atg agt 768 S   V   Y   G   S   T   V   G   H   T   I   I   D   L   M   S 256 aaaaat tgt tgg ggg ttg ttg gga cac tac ctg cgc gtc ctg atc cac 816 E   H   I   L   I   H   G   D   I   R   K   T   T   K   L   N 272 gagcac ata ttg att cac gga gat atc cgc aaa acc acc aaa ctg aac 864 I   G   G   T   E   I   E   V   E   T   L   V   E   D   E   A 288 atcggc gga acg gag atc gag gtc gag act ctc gtc gaa gac gaa gcc 912 I   G   G   T   E   I   E   V   E   T   L   V   E   D   E   A 304 gaggcc gga gcc gtg cca taa 933  E   A   G   A   V   P  stop 310

AAV Vector Injection

All of the animal experiments were at the institutional level and werein accord with the NIH Guide for the Care and Use of Laboratory Animals.

Newborn (PI) rat pups (Sprague-Dawley and Long-Evans) and mouse pups(C57BL/6J and C3H/HeJ or rd1/rd1) were anesthetized by chilling on ice.Adult mice (rd1/rd1) were anesthetized by IP injection of ketamine (100mg/kg) and xylazine (10 mg/kg). Under a dissecting microscope, anincision was made by scissors through the eyelid to expose the sclera. Asmall perforation was made in the sclera region posterior to the lenswith a needle and viral vector suspension of 0.8-1.5 μl at theconcentration of approximately 10¹¹ genomic particles/ml was injectedinto intravitreal space through the hole with a Hamilton syringe with a32-gauge blunt-ended needle. For each animal, usually only one eye wasinjected with viral vectors carrying Chop2-GFP and the other eye wasuninjected or injected with control viral vectors carrying GFP alone.After the injection, animals were kept on a 12/12 hr light/dark cycle.The light illumination of the room housing the animals measured at thewavelength of 500 nm was 6.0×10¹⁴ photons cm⁻²s⁻¹.

Histology

Animals were sacrificed at various time points after the vectorinjection. The expression of Chop2-GFP fluorescence was examined in flatwhole-mount retinas, vertical retinal, and coronal brain sections. Thedissected retinas and brains were fixed with 4% paraformaldehyde in PBSfor 0.5-2 hr at room temperature and 24 hr at 4° C., respectively. Thefixed retinas (embedded in 3% agarose) and brains were cut by using avibratome. The retinal and brain sections or the retinal whole mountswere mounted on slides and covered with Vectashield medium (VectorLaboratories). GFP fluorescence was visualized under a fluorescencemicroscope equipped with exciter, dichroic, and emission filters of465-495 nm, 505 nm, and 515-555 nm, respectively, and most images wereobtained with a digital camera (Axiocam, Zeiss). Some images wereobtained with a confocal microscope (TCS SP2, Leica). For lightmicroscopy of semithin vertical retinal section, eyes were enucleated,rinsed in PBS, and fixed in 1% osmium tetroxide, 2.5% glutaraldehyde,and 0.2 M Sorenson's phosphate buffer (pH 7.4) at 4° C. for 3 hr. Theeyes were then dehydrated in graded ethanols and embedded in plastic andcut into 1 μm sections and stained with a methylene blue/azure mixture.

Patch-Clamp Recordings

Dissociated retinal cells and retinal slice were prepared as previouslydescribed (Pan, 2000 and Cui et al., 2003). Recordings with patchelectrodes in the whole-cell configuration were made by an EPC-9amplifier and PULSE software (Heka Electronik, Lambrecht, Germany).Recordings were made in Hanks' solution containing (in mM): NaCl, 138;NaHCO₃, 1; Na₂HPO₄, 0.3; KCl, 5; KH₂PO₄, 0.3; CaCl₂, 1.25; MgSO₄, 0.5;MgCl₂, 0.5; HEPES-NaOH, 5; glucose, 22.2; with phenol red, 0.001% v/v;adjusted to pH 7.2 with 0.3 N NaOH.

The electrode solution contained (in mM): K-gluconate, 133; KCl, 7;MgCl₂, 4; EGTA, 0.1; HEPES, 10; Na-GTP, 0.5; and Na-ATP, 2; pH adjustedwith KOH to 7.4. The resistance of the electrode was 13 to 15 MΩ. Therecordings were performed at room temperature (˜22° C.).

Multielectrode Array Recordings

The multielectrode array recordings were based on the proceduresreported by Tian and Copenhagen (2003). Briefly, the retina wasdissected and placed photoreceptor side down on a nitrocellulose filterpaper strip (Millipore Corp., Bedford, Mass.). The mounted retina wasplaced in the MEA-60 multielectrode array recording chamber of 30 μmdiameter electrodes spaced 200 μm apart (Multi Channel System MCS GmbH,Reutlingen, Germany), with the ganglion cell layer facing the recordingelectrodes. The retina was continuously perfused in oxygenatedextracellular solution at 34° C. during all experiments. Theextracellular solution contained (in mM): NaCl, 124; KCl, 2.5; CaCl₂, 2;MgCl₂, 2; NaH₂PO₄, 1.25; NaHCO₃, 26; and glucose, 22 (pH 7.35 with 95%O₂ and 5% CO₂). Recordings were usually started 60 min after the retinawas positioned in the recording chamber. The interval between onsets ofeach light stimulus was 10-15 s. The signals were filtered between 200Hz (low cut off) and 20 kHz (high cut off). The responses fromindividual neurons were analyzed using Offline Sorter software (Plexon,Inc., Dallas, Tex.).

Visual-Evoked Potential Recordings

Visual-evoked potential recordings were carried out in wild-type mice ofthe C57BL/6 and 129/Sv strains aged 4-6 months and in the rd1/rd1 miceaged 6-11 months. Recordings were performed 2-6 months after viralvector injection.

After general anesthesia (i.p. injection of ketamine (100 mg/kg) andacepromazine (0.8 mg/kg), animals were mounted in a stereotaxicapparatus. Body temperature was either unregulated or maintained at 34°C. with a heating pad and a rectal probe. Pupils were dilated with 1%atropine and 2.5% accu-phenylephrine. A small portion of the skull(˜1.5×1.5 mm) centered about 2.5 mm from the midline and 1 mm rostral tothe lambdoid suture was drilled and removed. Recordings were made fromvisual cortex (area V1) by a glass micropipette (resistance ˜0.5 M afterfilling with 4 M NaCl) advanced 0.4 mm beneath the surface of the cortexat the contralateral side of the stimulated eye. The stimuli were 20 mspluses at 0.5 Hz. Responses were amplified (1,000 to 10,000), band-passfiltered (0.3-100 Hz), digitized (1 kHz), and averaged over 30-250trials.

Light Stimulation

For dissociated cell and retinal slice recordings, light stimuli weregenerated by a 150 W xenon lamp-based scanning monochromator withbandwidth of 10 nm (TILL Photonics, Germany) and coupled to themicroscope with an optical fiber. For multielectrode array recordings,light responses were evoked by the monochromator or a 175 W xenonlamp-based illuminator (Lambda LS, Sutter Instrument) with a band-passfilter of 400-580 nm and projected to the bottom of the recordingchamber through a liquid light guider. For visual evoked potential,light stimuli were generated by the monochromator and projected to theeyes through the optical fiber. The light intensity was attenuated byneutral density filters. The light energy was measured by a thin-typesensor (TQ82017) and an optical power meter (Model: TQ8210) (Advantest,Tokyo, Japan).

Example 2 Expression of Chop2 in Retinal Neurons In Vivo

To directly visualize the expression and localization of Chop2 proteins,the C-terminal portion of the Chop2 channel was replaced with GFP, tomake a Chop2-GFP chimera. The adeno-associated virus (AAV) vectors wasselected to target the expression of Chop2-GFP fusion protein intoretinal neurons because the capability of AAV vectors to delivertransgenes into nondividing cells, including inner retinal neurons(Harvey et al., 2002 and Martin et al., 2003), and to integrate thetransgenes into the host genome (Flotte, 2004).

A viral expression cassette, rAAV2-C AG-Chop2-GFP-WPRE, was made bysubcloning the Chop2-GFP chimera into an AAV serotype-2 expressioncassette containing a hybrid CMV enhancer/chicken β-actin (CAG) promoter(FIG. 1A). To establish the expression and function of Chop2 channels inretinal neurons in general, we first examined the expression of Chop2 innondystrophic retinas. The viral vector was injected into theintravitreal space in the eyes of postnatal day 1 rats and mice. Threeto four weeks after the injection, bright GFP fluorescence was observedin retinal neurons of all injected eyes (FIGS. 1B-1H), confirming thatChop2-GFP was expressed. The expression was usually confluent throughoutthe retina (FIG. 1B).

The Chop2-GFP-fluorescence was predominantly observed in retinalganglion cells (FIGS. 1C and 1D; also see FIG. 1H). The fluorescencesignal was observed throughout the inner plexiform layer (IPL) (FIG.1H), indicating that the viral vector targeted the expression ofChop2-GFP both in ON and OFF ganglion cells. The expressing of Chop2-GFPwas also frequently observed in horizontal cells (FIG. 1E), amacrinecells (FIG. 1F), and, occasionally, in bipolar cells (FIG. 1G).

The GFP signal was predominantly localized to the plasma membrane (FIG.1D), consistent with the GFP tag being anchored to the membrane by aseven-transmembrane portion of the Chop2 channel. Once expressed in acell, the GFP signal was extended over the entire cell including distalprocesses and axon terminals (see FIGS. 1C and 1E). Bright GFPfluorescence was found to be stable for 12 months or more after theinjection (FIG. 1H), whereas no gross changes in retinal morphology werenoticed (FIG. 1I). These results indicated that long-term stableexpression of Chop2-GFP was achieved in inner retinal neurons in vivo.

Example 3 Properties of Light-Evoked Currents of ChR2-Expressing InnerRetinal Neurons

Functional properties of the Chop2 channels were examined in innerretinal neurons by using whole-cell patch-clamp recordings. Therecordings were performed in acutely dissociated cells so thatphotoreceptor-mediated light responses were confidently excluded.Chop2-GFP-positive cells were identified by their GFP fluorescence (FIG.2A). The precursor for the Chop2 chromophore group, all-trans retinal,was not added because it might be ubiquitously present in cells (Kim etal, 1992 and Thompson and Gal, 2003). Light-evoked responses wereobserved in all recorded GFP fluorescent cells (n=34), indicating thatfunctional ChR2 (Chop2 with the chromophore attached) can be formed inretinal neurons with the retinal chromophore groups already present inthe cells. Consistently, the expression of functional ChR2 channels hasalso been recently reported in cultured hippocampal neurons without thesupply of exogenous retinal chromophore groups (Boyden et al, 2005; butsee Li et al, 2005).

The properties of the ChR2-mediated light responses were first examinedin voltage clamp. Light-evoked currents were observed inChop2-GFP-expressing inner retinal neurons by light stimuli up to thewavelength of 580 nm with the most sensitive wavelength around 460 nm(FIG. 2B), consistent with the reported peak spectrum sensitivity ofChR2 (Nagel et al, 2003). The amplitude and the kinetics of the currentswere dependent on the light intensity (FIG. 2C). FIGS. 2D and 2E show inthe expanded time scale the current traces right after the onset and thetermination of the light stimulation, respectively. Detectable currentswere observed in most recorded cells at a light intensity of 2.2×10¹⁵photons cm⁻²s⁻¹. In some cells, currents were observed at a lightintensity of 2×10¹⁴ photons cm⁻²s⁻¹ (not shown). At higher lightintensities, the currents displayed both transient and sustainedcomponents, similar to the properties of the nonfusion ChR2 (Nagel etal., 2003). The relationship between the light intensity and peakcurrent is shown in FIG. 2F (n=7). The activation and inactivationkinetics of the currents were also dependent on the light intensity(FIG. 2D). The initial phase of the current could be well fitted by anexponential function with a single activation and inactivation constant,as illustrated in FIG. 2D (red trace). The activation and inactivationtime constants versus light intensity are plotted in FIGS. 2G and 2H,respectively. On the other hand, the deactivation kinetics of thecurrents after the light off was not light-intensity dependent. Thecurrent decay trace could be well fitted by a single exponentialfunction as shown in FIG. 2E (red trace). The time constant was 17.1±6.5ms (mean±SD, n=7).

The next experiment examined whether the ChR2-mediated currents weresufficient to drive membrane depolarization. FIG. 3A shows therepresentative responses from a nonspiking neuron in response to fourincremental light intensities at the wavelength of 460 nm. Detectableresponses were observed in most recorded cells at a light intensity of2.2×10¹⁵ photons cm⁻²s⁻¹. At higher light intensities, the membranedepolarization approached a saturated level. The ChR2-mediated lightresponses to repeated light stimulations were further examined. Thetransient component of the currents diminished to repeated stimulationswhereas the sustained component of the currents was stable (top tracesin FIG. 3B). This was clearly seen in the expanded time scale in theright panel of FIG. 3B by comparing the superimposed first (red trace)and the second (black trace) light-evoked currents. For the same cell,in current clamp, the stimulations evoked robust membranedepolarizations (bottom traces in FIG. 3B). The membrane depolarizationsreached an almost identical level, except for the initial portion of theresponse. This was also shown in the expanded time scale (right panel),which superimposed the first (red trace) and the second (black trace)light-evoked responses. FIG. 3C shows a representative recording ofspiking neurons to repeated light stimulations. Again, the stimulationselicited almost identical membrane depolarizations accompanied bymultiple spikes. Taken together, these results demonstrated that theChR2-mediated currents in second- and third-order retinal neurons aresufficient to drive membrane depolarization and/or spike firing.

Example 4 Expression of Chop2 in Photoreceptor-Deficient rd1/rd1 Mice

Having established the expression and function of ChR2 in wild-typeretinas, we went on to address whether the expression of ChR2 couldrestore light responses in retinas after photoreceptor degeneration. Tothis end, the experiments were carried out in homozygous rd1 (rd1/rd1)mice (Bowes et al., 1990), a photoreceptor degeneration model with anull mutation in a cyclic GMP phosphodiesterase, PDE6, similar to someforms of retinitis pigmentosa in humans (McLaughlin et al., 1993). TheChop2-GFP viral vector was injected intravitreally into the eyes ofnewborn (P1) or adult mice at 2-12 months of age. Similar to the resultsobserved in wild-type animals, bright GFP signal was observed inChop2-GFP-injected retinas, predominately in retinal ganglion cells(FIGS. 4A and 4B). At the time of the recording experiments (≧4 monthsof age unless otherwise indicated), photoreceptor cells were absent(FIG. 4C). The expression of Chop2-GFP was observed in the rd1/rd1 miceup to 16 months of age (3-6 months after the viral injection) as thecase shown in FIG. 4A from a 15 month old rd1/rd1 mouse. These resultsindicate that inner retinal neurons in this photoreceptor-deficientmodel not only survive long after the complete death of photoreceptorsbut also retain the capability of stable expression of Chop2-GFP.

Example 5 Light-Evoked Responses of ChR2-Expressing Surviving InnerRetinal Neurons of rd1/rd1 Mice

The light response properties of the ChR2-expressing retinal neurons inrd1/rd1 mice were examined by whole-cell patch-clamp recording inretinal slices. The recordings were made from the GFP-positive cellslocated in the ganglion cell layer. Light-evoked currents were observedin GFP-positive cells. The magnitude of the current was again dependenton the light intensity (top traces in FIGS. 4D and 4E; also see lightintensity and current relationships shown in FIG. 4F). Two groups ofChR2-expressing retinal neurons were observed based on their responseproperties: a group of transient spiking neurons (FIG. 4D) and a groupof sustained spiking neurons (FIG. 4E). The membrane depolarizationand/or spike rates were also dependent on the light intensity (bottomtraces in FIGS. 4D and 4E). Furthermore, light at higher intensitiesmarkedly accelerated the kinetics of the voltage responses asillustrated in the right panels of FIGS. 4D and 4E by superimposing thesecond traces (black) and the fourth traces (red) in an expanded timescale. The relationships of light intensity to the membranedepolarization, the spike firing rate, and the time to the first spikepeak are shown in FIGS. 4G, 4H, and 4I, respectively. These resultsdemonstrate that the surviving retinal third-order neurons with theexpression of ChR2 are capable of encoding light intensity with membranedepolarization and/or action potential firing and response kinetics.

Example 6 Multielectrode Array Recordings of ChR2-Mediated RetinalActivities

The spike coding capability of the photoreceptor-deficient retina ofrd1/rd1 mice were examined after the expression of ChR2 by use ofmultielectrode array recordings from whole-mount retinas. As shown froma sample recording in FIG. 5A, spike firings with fast kinetics inresponse to light on and off were observed in Chop2-GFP-expressingretinas (n=11 retinas). The light-evoked spike firings were not affectedby the application of CNQX (25-50 μM) plus APV (25-50 μM) (n=3),indicating that the responses are originated from the ChR2 of therecorded cells. No such light-evoked spike firings were observed inretinas that were either injected with viral vectors carrying GFP alone(n=2 retinas) or left uninjected (n=3). The latter confirmed the absenceof photoreceptor-originated light responses. The light-evoked spikefirings were not affected by suramine (100 μM) (n=2), which has beenreported to be able to block melanopsin receptor-mediated photocurrent(Melyan et al., 2005 and Qiu et al., 2005).

In addition, the response kinetics to both light on and off (see FIG.5B) were much faster than those generated by the intrinsicallyphotosensitive retinal ganglion cells (Tu et al., 2005). These resultsindicated that a significant contribution to the observed lightresponses from the intrinsically photosensitive ganglion cells under ourrecording conditions is unlikely. The light-evoked responses were oftenfound to be picked up by the majority of the electrodes (see FIG. 5A),consistent with the observation that Chop2-GFP was extensively expressedin the retinas. The vast majority of the responses were sustained duringlight stimulation. FIG. 5B illustrates the raw traces recorded by asingle electrode in response to three incremental light stimuli. Theraster plots of the spike activity sorted from a single neuron of therecording were shown in FIG. 5C. The firing frequency was remarkablystable during the course of the recording. The averaged spike ratehistograms are shown in FIG. 5D. Again, the spike frequency wasincreased to the higher light intensity. The light responses could berecorded for up to 5 hr. These results demonstrate further that theChR2-expressing retinal ganglion cells can reliably encode lightintensity with spike firing rate.

Example 7 Visual-Evoked Potentials

A study was conducted to test whether the ChR2-mediated light responsesin the retinas of rd1/rd1 mice were transmitted to the visual cortex.The expression of transgenes, such as GFP, in retinal ganglion cells asachieved by AAV infection was reported to be able to extend to theirterminations in higher visual centers in the brain (Harvey et al.,2002). Therefore the anatomical projections of the axon terminals ofChop2-GFP-expressing retinal ganglion cells were first examined.Consistently, Chop2-GFP labeled axon terminals of retinal ganglion cellswere observed in several regions of the brain, including ventral lateralgeniculate nucleus and dorsal lateral geniculate nucleus (FIG. 6A), aswell as superior colliculus (FIG. 6B). These results indicate that thecentral projections of retinal ganglion cells in the degenerate retinasare maintained.

Visual evoked potentials (VEPs) from visual cortex were then examined.First, as illustrated in FIG. 6C, VEPs were observed in all testedwild-type mice (4-6 months of age) in response to light stimuli at thewavelengths of both 460 and 580 nm (n=6 eyes). When tested inChop2-GFP-injected eyes of rd1/rd1 mice (6-11 months of age), VEPs wereobserved in the majority of the eyes (nine out of 13) in response tolight stimulus at the wavelength of 460 nm but not to light stimulus atthe wavelength of 580 nm (FIG. 6D), consistent with the lightsensitivity of ChR2 channels (see FIG. 2B). The average amplitude of theVEPs in the Chop2-GFP-injected eyes in response to the light stimulus atthe wavelength of 460 nm was 110±34 μV (mean±SE; n=10), which is smallerthan that observed in wild-type mice (274±113 μV; n=6), although thesetwo values are not significantly different (one-way ANOVA test, p<0.1).The lower amplitudes of the VEPs in the Chop2-transfected mice comparedto the wild-type mice are not surprising because the expression of ChR2was probably only achieved in a small portion of the retinal ganglioncells. The average latency to the peak of the VEPs in theChop2-GFP-injected eyes was 45±1.7 ms (n=10), which is shorter than thatobserved in wild-type mice (62±2.8 ms; n=6). These two values weresignificantly different (p<0.01). The latter would be predicted becausethe light response mediated by ChR2 in retinal ganglion cells originatestwo synapses downstream of the photoreceptors. As a control, nodetectable VEPs were observed to light stimulus at the wavelength of 460nm in the eyes of the age-matched rd1/rd1 mice that were injected withviral vectors carrying GFP alone (n=5) (FIG. 6E). In addition, nodetectable VEPs were observed in uninjected rd1/rd1 mice (n=3; 5 monthsof age) to the wavelengths ranging from 420 to 620 nm (not shown),confirming that rd1/rd1 mice at ≧5 months of age are completely blindbased on VEPs.

To further ensure that the VEPs in the blind rd1/rd1 mice originate fromChR2 expressed in their retinas, the action spectrum of the VEP weremeasured by plotting their normalized amplitudes in response to varyinglight wavelengths and intensities to obtain the relative sensitivity ofthe response (FIG. 6F) (n=3). The data points were well fitted by avitamin-A₁-based visual pigment template (Partridge and De Grip, 1991)with a peak wavelength at 461 nm (FIG. 6G), a good match to the reportedpeak action spectrum of ChR2 at _(˜)460 nm (Nagel et al., 2003). Takentogether, these results demonstrated that expression of ChR2 in thephotoreceptor-deficient retinas can restore visually evoked responses inthe brain.

Example 8 Discussion of Examples 1-7

The results presented herein demonstrated that the strategy ofrestoration of light responses in photoreceptor-deficient rodent retinasbased on the expression of ChR2 is mechanistically and technicallyfeasible. Most importantly, the results showed that ChR2 satisfiesseveral major criteria for its use as a light sensor in retinal neurons.First, by delivery of an AAV vector carrying fused Chop2-GFP, theinventors showed the ability of retinal neurons to tolerate theprolonged expression of Chop2. To date, the expression of Chop2-GFPproteins had been achieved in nondystrophic rat retinal neurons for 12months and in photoreceptor deficient rd1/rd1 mice for 6 months in vivoafter the viral injection. The present results therefore indicate thatthe expression of ChR2 in retinal neurons is biocompatible under normallight cycle conditions.

Second, these results showed that a sufficient number of ChR2 can beformed in retinal neurons, with only endogenous chromophore groups assupplied by regular diet, to produce robust membrane depolarizationsand/or action potential firings in the retina and VEPs in visual cortex.It is worth emphasizing here that, unlike animal visual pigments thatrapidly lose their chromophore after its photoisomerization from 11-cisto all-trans retinal (Wald, 1968), for microbial-type rhodopsins,photoisomerization from all-trans to 11-cis retinal is reversible andboth isomers remain attached to the protein (Oesterhelt, 1998). Once theChR2 complex is formed, the light-sensitive channel can sustain multiplecycles of photoisomerization with the same chromophore moiety. Althoughthe efficacy of the de novo ChR2 formation might be expected to dependon the availability of the chromophore group, the need for constantresupply of the chromophore to form new ChR2 does not appear to impose alimitation on overall ChR2 function. As observed in the multielectrodearray recordings, ChR2 respond repeatedly to light stimulation forseveral hours in vitro without loss of activity. These results thusindicate that the turn-over rate for ChR2 is fairly slow, an additionaladvantage for use as an artificially produced light sensor.

Furthermore, as reported originally in cell expression systems (Nagel etal., 2003), later in hippocampal neurons (Boyden et al., 2005, Ishizukaet al., 2006 and Li et al., 2005), and now shown in retinal neurons, anumber of properties of the ChR2 channel are highly advantageous for itsuse as a light sensor.

First, the ChR2 channel is permeable to the cations that underlieneuronal membrane excitability. Thus, activation of ChR2 channels bylight can directly produce membrane depolarizations to mimic theON-responses of inner retinal neurons. Indeed, as shown herein, thelight-evoked responses mediated by ChR2 in nonspiking and spikingretinal neurons remarkably resemble the light responses of ON-bipolarcells and sustained ON-ganglion cells (Werblin and Dowling, 1969 andKaneko, 1970).

Second, the activation kinetics of the current in response to light areextremely fast, whereas the sustained components of the currents do notshow apparent inactivation to continuous or repeated lightilluminations. Thus, the ChR2-expressing neurons can signal with rapidkinetics but without pigment inactivation. Consistently, the expressionof ChR2 has been shown to allow optical control of neural excitabilitywith high temporal resolution (Boyden et al., 2005, Ishizuka et al.,2006 and Li et al., 2005). Furthermore, it is shown here that themagnitude and activation kinetics of the light-evoked current dependupon light irradiance over a 3-log-unit range. As demonstrated in thewhole-cell and multielectrode array recordings, this would allow theencoding of various light intensities with graded membranedepolarizations and/or spike rates.

Also of importance for the feasibility of the strategy of restoringlight sensitivity in retinas after photoreceptor degeneration, resultsof this study show that many inner retinal neurons survive in agedrd1/rd1 mice (up to 16 months of age) and are capable of expressing ChR2long after the death of all photoreceptors. This is consistent withhistological studies showing that many inner retinal neurons survive,despite some remodeling, in this mouse model (Jimenez et al., 1996,Strettoi and Pignatelli, 2000 and Chang et al., 2002). Moreover, thepresent studies using ChR2 showed that the surviving inner retinalneurons retained their physiological capability to encode light signalswith membrane depolarizations and/or action potential firings and totransmit visual signals to the visual cortex. Thus, the strategy basedon the expression of ChR2 is suitable at least for certain retinaldegenerative diseases at certain stages.

The remodeling of inner retinal neurons triggered by photoreceptordegeneration raised some concerns for the retinal-based rescue strategyafter the death of photoreceptors (Strettoi and Pignatelli, 2000, Joneset al., 2003 and Jones and Marc, 2005). However, retinal degenerativediseases are heterogeneous as to the time course of the degeneration,survival an d functional state of different cell types (Chang et al.,2002). The use of ChR2 is a powerful tool for undertaking such studies.

Retinal remodeling is believed to be caused by deafferentation (Jonesand Marc, 2005). Therefore, the restoration of the light sensitivity ininner retinal neurons may be able to prevent or delay the remodelingprocesses.

Finally, according to the present invention, viral-based gene deliverysystems, such as AAV vectors (Flannery et al., 1997, Bennett et al.,1999, Ali et al., 2000 and Acland et al., 2001), are tools forintroducing Chop2 into retinal neurons as demonstrated herein.

The present results showed that that viral construct with AAV serotype-2and CAG promoter achieved robust expression of Chop2 in ganglion cells.However, because the expression of Chop2 with this construct appears totarget both ON- and OFF-type ganglion cells, it remains to be determinedhow the conversion of both ON- and OFF-ganglion cells into ON-typeaffects the visual perception.

Behavior studies in primates reported that pharmacological blockade ofthe ON channel in the retina did not severely impair such visionfunctions as the detection of light decrement and the perception ofshape (Schiller et al., 1986). Therefore, targeting of ChR2 to the ONchannel, for example to ON-type ganglion cells, is expected to result inuseful vision.

It is also contemplated herein to express ChR2 in the more distalretinal neurons, such as bipolar cells; this approach would utilize theremaining signal processing functions of the degenerate retina.Targeting ChR2 to rod bipolar cells is particularly attractive becausethe depolarization of rod bipolar cells can lead to the ON and OFFresponses at the levels of cone bipolar cells and retinal ganglion cells(Wassle, 2004), thereby maintaining the ON and OFF channels that areinherent in the retina.

The threshold light intensity required for producing responses inChR2-expressing retinas appeared to be near 10¹⁴-10¹⁵ photons cm⁻²s⁻¹.For comparison, the thresholds for normal rod and cone photoreceptorsare about 10⁶ and 10¹⁰ photons cm⁻²s⁻¹, respectively (Dacey et al.,2005). Therefore, the ChR2-expressing retinas would operate insubstantially higher photonic range. The relatively low lightsensitivity of the ChR2-expressing retinas compared to the normalretinas could be due to a number of factors. First, there may be a lowcross-sectional density of ChR2 molecules in the transfected retinalneurons compared with the visual pigments in rods and cones. Second, theChR2-expressing inner retinal neurons lack the unique multilayerphotoreceptor membrane organization, typical for the outer segments ofrods and cones, which developed to achieve higher pigment density andthus increase the probability of catching photons (Steinberg, et al.,1980). Third, unlike visual pigments that propagate their signal throughamplification cascade (Stryer, 1991), the directly light-gated ChR2channels lack such amplification capabilities. Finally, in normalretinas, amplification of visual signals occurs as the signals convergefrom multiple photoreceptors to ganglion cells (Barlow et al., 1971).This process was not yet achieved in the ChR2-transfected retinas. It isnot yet evident which of these factors contributes the most to thedecreased light sensitivity of the ChR2-expressing retinas remains.Interestingly, ChR2 mediated phototaxis to low-intensity light in greenalgae (Sineshchekov et al., 2002; but see Kateriya et al. [2004]).Therefore, the light sensitivity of ChR2 in retinal neurons may havebeen altered by modifications introduced in the Chop2 molecule for theheterologous expression. Such a difference may also reflect differentstructural and functional organization of algae and mammalian cells.

Nevertheless, for clinical usage, light intensifying devices can be usedto expand the light operation range.

At present, no treatment is available for restoring vision once thephotoreceptor cells have been lost. As noted above, transplantation ofnormal photoreceptor cells or progenitor cells (Bok, 1993 and Lund etal., 2001) or direct electrical stimulation of the surviving second- andthird-order retinal neurons via retinal implants (Zrenner, 2002) havebeen proposed as possible strategies for restoration of light responsesin the retina after rod and cone degeneration. An important advantage ofthe present invention is that it does not involve the introduction oftissues or devices into the retina and, therefore, may largely avoid thecomplications of immune reactions and bioincompatibilities. In addition,the present approach is expected to achieve high spatial resolution forthe restored “vision” because the approach targets the cellular level.Thus, the expression of microbial-type channel rhodopsins, such as ChR2,in surviving retinal neurons is a strategy for the treatment of completeblindness caused by rod and cone degeneration.

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All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by thoseskilled in the art in light of the teachings and guidance presentedherein, in combination with the knowledge of one of ordinary skill inthe art.

1. A method of restoring light sensitivity to a retina, comprising: (a)delivering to retinal neurons a nucleic acid expression vector thatencodes a light-gated channel rhodopsin or a light-driven ion pumprhodopsin expressible in said retinal neurons, which vector comprises anopen reading frame encoding the rhodopsin, and operatively linkedthereto, a promoter sequence, and optionally, transcriptional regulatorysequences; and (b) expressing said vector in said retinal neurons,thereby restoring the light sensitivity.
 2. The method of claim 1wherein the rhodopsin is channelrhodopsin-2 (Chop2) the sequence ofwhich is SEQ ID NO:6, or a biologically active fragment thereof thatretains the biological activity of Chop2 or is a biologically activeconservative amino acid substitution variant of SEQ ID NO:6 or of saidfragment.
 3. The method of claim 1 wherein the vector is a rAAV viralvector.
 4. The method of claim 1 wherein the promoter is a constitutivepromoter.
 5. The method of claim 4 wherein the constitutive promoter isa CMV promoter or a hybrid CMV enhancer/chicken β-actin (CAG) promoter.6.-7. (canceled)
 8. The method of claim 1 wherein the promoter is aninducible and/or a cell type-specific promoter.
 9. The method of claim 8wherein the cell type-specific promoter is selected from the groupconsisting of a mGluR6 promoter, a Pcp2 (L7) promoter or a neurokinin-3(NK-3) promoter.
 10. The method of claim 9 wherein the promoter is themGlu6 promoter and is part of a promoter sequence SEQ ID NO:7. 11.(canceled)
 12. The method of claim 2 wherein the vector comprises a CAGpromoter, a woodchuck posttranscriptional regulatory element (WPRE), anda human or bovine growth hormone polyadenylation sequence.
 13. Themethod of claim 1 wherein the retinal neurons are ON-type retinalganglion cells, OFF-type retinal ganglion cells, retinal rod bipolarcells, AII amacrine cells. ON retinal cone bipolar cells and OFF retinalcone bipolar cells. 14.-16. (canceled)
 17. The method of claim 9 whereinthe promoter is an NK-3 promoter and the vector is delivered to andexpressed in OFF retinal cone bipolar cells.
 18. A method of restoringphotosensitivity to a retina of a subject suffering from vision loss orblindness in whom retinal photoreceptor cells are degenerating or havedegenerated and died, which method comprises: (a) delivering to theretina of said subject a nucleic acid vector that encodes a light-gatedchannel rhodopsin or a light-driven ion pump rhodopsin expressible inretinal neurons; which vector comprises an open reading frame encodingthe rhodopsin, and operatively linked thereto, a promoter sequence, andoptionally, transcriptional regulatory sequences; (b) expressing saidvector in said retinal neurons, wherein the expression of the rhodopsinrenders said retinal neurons photosensitive, thereby restoringphotosensitivity to said retina.
 19. The method of claim 18 wherein therhodopsin is Chop2 or a biologically active fragment or conservativeamino acid substitution variant thereof.
 20. The method of claim 18wherein the vector is a rAAV viral vector.
 21. The method of claim 18wherein the promoter is a constitutive promoter.
 22. The method of claim21 wherein the constitutive promoter is a CMV promoter or a hybrid CAGpromoter. 23.-24. (canceled)
 25. The method of claim 18 wherein thepromoter is an inducible or a cell type-specific promoter.
 26. Themethod of claim 25 wherein the cell type-specific promoter is selectedfrom the group consisting of a mGluR6 promoter, a Pcp2 (L7) promoter ora neurokinin-3 (NK-3) promoter.
 27. The method of claim 26 wherein thepromoter is the mGlu6 promoter and is part of a promoter sequence SEQ IDNO:7.
 28. The method of claim 18 wherein the retinal neurons are ON-typeretinal ganglion cells, OFF-type retinal ganglion cells, retinal rodbipolar cells, AII amacrine cells, ON-type retinal cone bipolar cells orOFF-type retinal cone bipolar cells. 29.-31. (canceled)
 32. The methodof claim 26 wherein the promoter is the NK-3 promoter and the vector istargeted to OFF cone bipolar cells.
 33. The method of claim 18 whereinthe restoration of photosensitivity results in restoration of vision insaid subject, as measured by one of more of the following methods: (i) alight detection response by the subject after exposure to a lightstimulus (ii) a light projection response by the subject after exposureto a light stimulus; (iii) light resolution by the subject of a lightversus a dark patterned visual stimulus; (iv) electrical recording of aresponse in the visual cortex to a light flash stimulus or a patternvisual stimulus.
 34. (canceled)
 35. The method of claim 18 wherein saidvision loss or blindness is a result of a degenerative disease.
 36. Themethod of claim 35 wherein said disease is retinitis pigmentosa orage-related macular degeneration.
 37. The method of claim 18 wherein thesubject is also provided with a visual prosthesis before, at the sametime as, or after delivery of said vector, which prosthesis is a retinalimplant, a cortical implant, a lateral geniculate nucleus implant, or anoptic nerve implant.
 38. (canceled)
 39. The method of claim 18 whereinthe subject's visual response is subjected to training using one or morevisual stimuli.
 40. (canceled)
 41. The method of claim 39 wherein saidtraining is achieved by one of more of the following methods: (a)habituation training characterized by training the subject to recognize(i) varying levels of light and/or pattern stimulation, and/or (ii)environmental stimulation from a common light source or object; and (b)orientation and mobility training characterized by training the subjectto detect visually local objects and move among said objects moreeffectively than without the training.
 42. The method of claim 2 whereinthe amino acid sequence of the biologically active fragment is SEQ IDNO:3, or is a conservative amino acid substitution variant thereof thatretains the biological activity of said Chop2 fragment.